CA3193753A1

CA3193753A1 - Compositions and methods for treatment of duchenne muscular dystrophy

Info

Publication number: CA3193753A1
Application number: CA3193753A
Authority: CA
Inventors: Jesper Gromada; Tudor FULGA; Alison MCVIE-WYLIE; Giselle DOMINGUEZ GUTIERREZ; Yurong XIN; Yi-Li Min; Mehmet Fatih BOLUKBASI; Eric Anderson
Original assignee: Vertex Pharmaceuticals Inc
Current assignee: Vertex Pharmaceuticals Inc
Priority date: 2020-09-09
Filing date: 2021-09-08
Publication date: 2022-03-17
Also published as: AU2021342057A1; MX2023002755A; EP4211242A1; KR20230084491A; JP2023540783A; IL300949A

Abstract

Compositions and methods for treating Duchenne Muscular Dystrophy (DMD) are encompassed.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULAR
DYSTROPHY
[0001] This application claims the benefit of priority to United States Provisional Application No. 63/076,250, filed September 9, 2020; United States Provisional Application No. 63/152,114, filed February 22, 2021; United States Provisional Patent Application No.
63/166,174, filed March 25, 2021; and United States Provisional Application No. 63/179,850, filed April 26, 2021; all of which are incorporated by reference in their entirety.

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 7, 2021, is named 2021-09-07 01245-0024-00PCT

5T25.txt and is 646,734 bytes in size.
INTRODUCTION AND SUMMARY

[0003] Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Duchenne muscular dystrophy (DMD) is one of the most severe forms of MD that affects approximately 1 in 5000 boys and is characterized by progressive muscle weakness and premature death.
Cardiomyopathy and heart failure are common, incurable and lethal features of DMD. The disease is caused by mutations in the gene encoding dystrophin (DMD), which result in loss of expression of dystrophin, causing muscle membrane fragility and progressive muscle wasting.

[0004] CRISPR-based genome editing can provide sequence-specific cleavage of genomic DNA
using a Cas9 and a guide RNA. For example, a nucleic acid encoding the Cas9 enzyme and a nucleic acid encoding for the appropriate guide RNA can be provided on separate vectors or together on a single vector and administered in vivo or in vitro to knockout or correct a genetic mutation, for example. The approximately 20 nucleotides at the 5' end of the guide RNA
serves as the guide or spacer sequence that can be any sequence complementary to one strand of a genomic target location that has an adjacent protospacer adjacent motif (PAM). The PAM sequence is a short sequence adjacent to the Cas9 nuclease cut site that the Cas9 molecule requires for appropriate binding. The nucleotides 3' of the guide or spacer sequence of the guide RNA serve as a scaffold sequence for interacting with Cas9. When a guide RNA and a Cas9 are expressed, the guide RNA will bind to Cas9 and direct it to the sequence complementary to the guide sequence, where it will then initiate a double-stranded break (DSB). To repair these breaks, cells typically use an error prone mechanism of non-homologous end joining (NHEJ) which can lead to disruption of function in the target gene through insertions or deletion of codons, shifts in the reading frame, or result in a premature stop codon triggering nonsense-mediated decay. See, e.g., Kumar et al. (2018) Front. Mol. Neurosci. Vol.
11, Article 413.

[0005] While gene editing strategies using systems (e.g., CRISPR) for treating DMD have been previously explored, these strategies have focused primarily on either: a) cutting at multiple different sites to excise large portions (e.g., one or more exons) of the dystrophin gene (see, e.g., Ousterout et al., 2015, Nat Commun. 6:6244); or b) cutting in a single site to introduce indels that either result in a frame-shifting mutation and/or that destroy a splice acceptor/donor site in the dystrophin gene (see, e.g., Amoassi et al., 2018, Science, 362(6410):86-91). However, there remains a need for additional alternative and effective gene editing strategies for treating diseases like DMD.

[0006] In order for gene editing systems like CRISPR to be effective in treating diseases and disorders like DMD in a patient, effective delivery vectors of the CRISPR
system components are necessary. Adeno-associated virus (AAV) administration of the CRISPR-Cas components in vivo or in vitro is attractive due to the early and ongoing successes of AAV vector design, manufacturing, and clinical stage administration for gene therapy. See, e.g., Wang et al. (2019) Nature Reviews Drug Discovery 18:358-378; Ran et al. (2015a) Nature 520: 186-101. However, the commonly used Streptococcus pyogenes (spCas9) is very large, and when used in AAV-based CRISPR/Cas systems, requires two AAV vectors ¨ one vector carrying the nucleic acid encoding the spCas9, and the other carrying the nucleic acid encoding the guide RNA. One possible way to overcome this technical hurdle is to take advantage of the smaller orthologs of Cas9 derived from different prokaryotic species. Smaller Cas9's such as Staphylococcus aureus (SaCas9) and Staphylococcus lugdunensis (SluCas9) may be able to be manufactured on a single AAV vector together with a nucleic acid encoding one or more guide RNAs. One advantage of incorporating one or more guide RNAs on a single vector together with the smaller SaCas9 or SluCas9 is that doing so allows extreme design flexibility in situations where more than one guide RNA is desired for optimal performance. For example, one vector may be utilized to express SaCas9 or SluCas9 and one or more guide RNAs targeting a first genomic target (e.g., a pair of guide RNAs that together bind regions flanking a genomic target), and a second vector may be utilized to express multiple copies of the same (e.g., the same pair of guides in the first vector) or different guide RNAs targeting the same or a different genomic target. As another example, multiple copies of the same guide RNA may be beneficial, and the use of smaller Cas9's allow for multiple copies of guide RNA to be incorporated on the same vector as the Cas9, and also for even more copies of guide RNA when combined with a second vector. Compositions and methods utilizing these configurations have the benefit of reducing manufacturing costs, reducing complexity of administration routes and protocols, and allowing maximum flexibility with regard to using multiple copies of the same or different guide RNAs targeting the same or different genomic target sequences. In some instances, providing multiple copies of the same guide RNA improves the efficiency of the guide, improving an already successful system.

[0007] Provided herein are compositions and methods for treating DMD utilizing the smaller Cas9s from Staphylococcus aureus (SaCas9) and Staphylococcus lugdunensis (SluCas9).

Compositions comprising a single AAV vector comprising a nucleic acid molecule encoding SaCas9 or SluCas9 and a guide RNA are provided.

[0008] Accordingly, the following non-limiting embodiments are provided.
Embodiment Al is a composition comprising:
a. a single nucleic acid molecule comprising:
i. a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; or ii. a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii. a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and 1, 2, or 3 guide RNAs; or b. two nucleic acid molecules comprising:
i. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9); and a second nucleic acid that does not encode a SaCas9 or SluCas9 and encodes any one of the following:
1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or 2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid;
or 3. from one to six guide RNAs; or ii. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and 1. at least one, at least two, or at least three guide RNAs; or 2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid;
or 3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SaCas9 or SluCas9, optionally wherein the second nucleic acid comprises any one of the following:
1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or 2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid;
or 3. from one to six guide RNAs; or iii. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SaCas9 or SluCas9 and encodes from one to six guide RNAs; or iv. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and at least two guide RNAs, wherein at least one guide RNA binds upstream of a target sequence and at least one guide RNA binds downstream of the target sequence; and a second nucleic acid that does not encode a SaCas9 or SluCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid, wherein the guide RNA(s) target a region in the dystrophin gene.
Embodiment A2 is a composition comprising two nucleic acid molecules comprising i) a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and a first and a second guide RNA that function to excise a portion of a DMD
gene; and ii) a second nucleic acid encoding at least 2 or at least 3 copies of the first guide RNA and at least 2 or at least 3 copies of the second guide RNA.
Embodiment A3 is a composition comprising one or more nucleic acid molecules encoding an endonuclease and a pair of guide RNAs, wherein each guide RNA targets a different sequence in a DMD gene, wherein the endonuclease and pair of guide RNAs are capable of excising a DNA fragment from the DMD gene; wherein the DNA fragment is between 5-250 nucleotides in length.

Embodiment A4 is the composition of claim 3, wherein the endonuclease is a class 2, type II Cas endonuclease.
Embodiment A5 is the composition of claim 3, wherein the class 2, type II Cas endonuclease is SpCas9, SaCas9, or SluCas9.
Embodiment A6 is the composition of claim 3, wherein the endonuclease is not a class 2, type V
Cas endonuclease.
Embodiment A7 is the composition of claim 3, wherein the excised DNA fragment comprises a splice acceptor site or a splice donor site.
Embodiment A8 is the composition of claim 3, wherein the excised DNA fragment comprises a premature stop codon in the DMD gene.
Embodiment A9 is the composition of claim 3, wherein the excised DNA fragment does not comprise an entire exon of the DMD gene.
Embodiment A10 is the composition of any one of claims 1-9, wherein the guide RNA comprises any one of the following:
a. when SaCas9 is used, one or more spacer sequences selected from any one of SEQ ID
NOs: 1-35, 1000-1078, and 3000-3069; or b. when SluCas9a is used, one or more spacer sequences selected from any one of SEQ
ID NOs: 100-225, 2000-2116, and 4000-4251; or c. when SaCas9 is used, one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:
1-35, 1000-1078, and 3000-3069; or d. when SluCas9a is used, one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:
100-225, 2000-2116, and 4000-4251; or e. when SaCas9 is used, one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; or f. when SluCas9 is used, one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; or g. when SaCas9 is used with at least two guide RNAs, a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ
ID
NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16;

1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016;
1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12;
1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; or h. when SaCas9 is used with at least two guide RNAs, at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10;
1005 and 1017; 16 and 1017; and 16 and 1018; or i. when SaCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10;
16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16;
1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017;
and 16 and 1018; or j. when SluCas9 is used with at least two guide RNAs, a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ
ID
NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or k. when SluCas9 is used with at least two guide RNAs, at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135;

150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131;

and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or 1. when SluCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or m. when SluCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150; and v. SEQ ID Nos: 146 and 148; or n. when a SaCas9-KKH is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016.
Embodiment All is a composition comprising a single nucleic acid molecule encoding one or more guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises:
a. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or b. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or c. a first nucleic acid encoding one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:
1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or d. a first nucleic acid encoding one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:
100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or e. a first nucleic acid encoding one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or f. a first nucleic acid encoding one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or g. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12;
1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or h. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16;
12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003;
12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16;
1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or i. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005;
1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012;
12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10;

and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003;

and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or j. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136;
139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149;

and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or k. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or 1. a first nucleic acid encoding a pair of guide RNAs that is at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or m. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;

9 iv. SEQ ID Nos: 144 and 150;
v. SEQ ID Nos: 146 and 148;
and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or n. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016;
iii. and a second nucleic acid encoding a SaCas9-KKH.
Embodiment Al2 is a composition comprising one or more nucleic acid molecules encoding a Staphylococcus lugdunensis (SluCas9) and at least two guide RNAs, wherein a first and second guide RNA target different sequences in a DMD gene, wherein the first and a second guide RNA comprise a sequence that is at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150;
v. SEQ ID Nos: 146 and 148.
Embodiment A13 is a composition comprising one or more nucleic acid molecules encoding an endonuclease and at least two guide RNAs, wherein the guide RNAs each target a different sequence in a DMD gene, wherein the guide RNAs each comprise a sequence that is at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016; and b. a second nucleic acid encoding a SaCas9-KKH.
Embodiment A14 is the composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least two guide RNAs.
Embodiment A15 is the composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least three guide RNAs.

Embodiment A16 is the composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least four guide RNAs.
Embodiment A17 is the composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least five guide RNAs.
Embodiment A18 is the composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least six guide RNAs.
Embodiment A19 is the composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least seven guide RNAs.
Embodiment A20 is the composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least eight guide RNAs.
Embodiment A21 is the composition of any one of the preceding claims, wherein the first nucleic acid comprises a nucleotide sequence encoding an endonuclease and at least one, at least two, or at least three guide RNAs.
Embodiment A22 is the composition of any one of the preceding claims, wherein the first nucleic acid comprises a nucleotide sequence encoding an endonuclease and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid.
Embodiment A23 is the composition of any one of the preceding claims, wherein the first nucleic acid comprises a nucleotide sequence encoding an endonuclease and from one to three guide RNAs.
Embodiment A24 is the composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs.
Embodiment A25 is the composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid.
Embodiment A26 is the composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes from one to six guide RNAs.
Embodiment A27 is the composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 guide RNAs.
Embodiment A28 is the composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes 2, 3, 4, 5, or 6 guide RNAs.

Embodiment A29 is the composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the guide RNA encoded by the first nucleic acid and the second nucleic acid are the same.
Embodiment A30 is the composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the guide RNA encoded by the first nucleic acid and the second nucleic acid are different.
Embodiment A31 is the composition of any one of the preceding claims, comprising at least two nucleic acid molecules encoding at least two guide RNAs, wherein at least one guide RNA
binds to a target sequence within an exon in the DMD gene that is upstream of a premature stop codon, and wherein at least one guide RNA binds to a target sequence within an exon in the DMD gene that is downstream of a premature stop codon.
Embodiment A32 is the composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the first nucleic acid molecule and the second nucleic acid molecule each encode the same guide RNA.
Embodiment A33 is the composition of any one of the preceding claims, comprising at least two nucleic acid molecules each encoding at least one guide RNA, wherein the second nucleic acid molecule encodes a guide RNA that binds to the same target sequence as the guide RNA
in the first nucleic acid molecule.
Embodiment A34 is the composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the second nucleic acid molecule encodes at least 2, at least 3, at least 4, at least 5, or at least 6 guide RNAs, wherein the guide RNAs in the second nucleic acid molecule bind to the same target sequence as the guide RNA in the first nucleic acid molecule.
Embodiment A35 is the composition of any one of the preceding claims, wherein the composition comprises at least two nucleic acid molecules, wherein the first nucleic acid molecule comprises a sequence encoding an endonuclease, wherein the second nucleic acid molecule encodes a first guide RNA and a second guide RNA, wherein the first guide RNA
and the second guide RNA are not the same sequence, and wherein the second nucleic acid molecule does not encode an endonuclease.
Embodiment A36 is the composition of claim 35, wherein the first nucleic acid molecule also encodes a copy of the first guide RNA and a copy of the second guide RNA.
Embodiment A37 is the composition of claim 35 or 36, wherein the second nucleic acid molecule encodes two copies of the first guide RNA and two copies of the second guide RNA.

Embodiment A38 is the composition of any one of claims 35-37, wherein the second nucleic acid molecule encodes three copies of the first guide RNA and three copies of the second guide RNA.
Embodiment A39 is the composition of any one of claims 35-37, wherein the first nucleic acid molecule comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence, the reverse complement of a nucleotide sequence encoding the first guide RNA sequence, the reverse complement of a promoter for expression of the nucleotide sequence encoding the first guide RNA sequence, a promoter for expression of a nucleotide sequence encoding the endonuclease, a nucleotide sequence encoding an endonuclease, a polyadenylation sequence, a promoter for expression of the second guide RNA in the same direction as the promoter for the endonuclease, the second guide RNA
sequence, and a second guide RNA scaffold sequence.
Embodiment A40 is the composition of claim 39, wherein the promoter for expression of the nucleotide sequence encoding the first guide RNA sequence in the first nucleic acid molecule is a U6 promoter and the promoter for expression of the nucleotide sequence encoding the second guide RNA in the first nucleic acid molecule is a U6 promoter.
Embodiment A41 is the composition of any one of claims 35-40, wherein the first nucleic acid molecule is in a first vector, and wherein the second nucleic acid is in a separate second vector.
Embodiment A42 is the composition of any one of claims 35-41, wherein the first nucleic acid molecule encodes a Staphylococcus aureus Cas9 (SaCas9) endonuclease, and wherein the first guide RNA comprises the first sequence and the second guide RNAs comprises the second sequence from any one the following pairs of sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003;
12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018.
Embodiment A43 is the composition of claim 42, wherein the first guide RNA
comprises the sequence of SEQ ID NO: 12 and the second guide RNA comprises the sequence of SEQ ID
NO: 1013.
Embodiment A44 is the composition of any one of claims 35-43, wherein the first nucleic acid molecule encodes a Staphylococcus lugdunensis (SluCas9) endonuclease, and wherein the first guide RNA comprises the first sequence and the second guide RNAs comprises the second sequence from any one the following pairs of sequences: SEQ ID NOs: 148 and 134;

149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151;
140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139;
151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144;
131 and 145; 151 and 145; and 148 and 146.
Embodiment A45 is the composition of claim 44, wherein:
i. the first guide RNA comprises the sequence of SEQ ID NO: 148 and the second guide RNA comprises the sequence of SEQ ID NO: 134; or ii. the second guide RNA comprises the sequence of SEQ ID NO: 145 and the second guide RNA comprises the sequence of SEQ ID NO: 131.
Embodiment A46 is the composition of any one of claims 35-45, wherein the first nucleic acid is in a first vector, and wherein the second nucleic acid is in a separate second vector.
Embodiment A47 is the composition of claim 46, wherein the first and second vectors are viral vectors.
Embodiment A48 is the composition of claim 47, wherein the viral vectors are AAV9 vectors.
Embodiment A49 is the composition of claim 48, wherein the AAV9 vectors are each less than 5kb, less than 4.9kb, less than 4.85, less than 4.8, or less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs.
Embodiment A50 is the composition of claim 48 or 49, wherein the AAV9 vectors are each between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs.
Embodiment A51 is the composition of any one of claims 47-50, wherein the first vector is between 4.4 - 4.85 kb from ITR to ITR in size, inclusive of both ITRs.
Embodiment A52 is the composition of any one of the preceding claims, wherein the guide RNA
binds to one or more target sequences within a DMD gene.
Embodiment A53 is the composition of any one of the preceding claims, wherein the guide RNA
binds to one or more target sequences within an exon of the DMD gene.
Embodiment A54 is the composition of any one of the preceding claims, comprising two guide RNAs, wherein i) each guide RNA targets a sequence within an exon; ii) one guide RNA
targets a sequence within an exon and one targets a sequence within an intron;
or iii) each guide RNA targets a sequence within an intron.

Embodiment A55 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein i) each guide RNA targets the same genomic target sequence; ii) each guide RNA targets a different target sequence; or iii) at least one guide RNA
targets one sequence and at least one guide RNA targets a different sequence.
Embodiment A56 is the composition of any one of the preceding claims, comprising a guide RNA that binds to an exon of the DMD gene, wherein the exon is selected from exon 43, 44, 45, 50, 51, and 53.
Embodiment A57 is the composition of any one of the preceding claims, comprising at least two guide RNAs that binds to an exon of the DMD gene, wherein at least one guide RNA binds to a target sequence within an exon in the DMD gene, and at least one guide RNA
binds to a different target sequence within the same exon in the DMD gene.
Embodiment A58 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein at least one guide RNA binds to a target sequence within an exon that is upstream of a premature stop codon, and at least one guide RNA binds to a target sequence within an exon that is downstream of a premature stop codon.
Embodiment A59 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein at least one guide RNA binds to a target sequence within an exon in the DMD gene, and at least one guide RNA binds to a different target sequence within the same exon in the DMD gene, wherein when expressed in vivo or in vivo, a portion of the exon is excised.
Embodiment A60 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon.
Embodiment A61 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, and wherein the portion of the exon remaining after excision are rejoined with a one nucleotide insertion.
Embodiment A62 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, wherein the portion of the exon remaining after excision is rejoined without a nucleotide insertion.
Embodiment A63 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, wherein the size of the excised portion of the exon is between 5 and 250 nucleotides in length.
Embodiment A64 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, wherein the size of the excised portion of the exon is between 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 75, 5 and 50, 5 and 25, 5 and 10, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 75, 20 and 50, 20 and 25, 50 and 250, 50 and 200, 50 and 150, 50 and 100, and 50 and 75 nucleotides.
Embodiment A65 is the composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclesae, the guide RNAs excise a portion of the exon, wherein the size of the excised portion of the exon is between 8 and 167 nucleotides.
Embodiment A66 is the composition of any one of the preceding claims, wherein the guide RNA
is an sgRNA.
Embodiment A67 is the composition of any one of the preceding claims, wherein the guide RNA
is modified.
Embodiment A68 is the composition of any one of the preceding claims, wherein the guide RNA
is modified, and wherein the modification alters one or more 2' positions and/or phosphodiester linkages.
Embodiment A69 is the composition of any one of the preceding claims, wherein the guide RNA
is modified, and wherein the modification alters one or more, or all, of the first three nucleotides of the guide RNA and/or the last three nucleotides of the sgRNA.
Embodiment A70 is the composition of any one of claims 66-68, wherein the modification alters one or more, or all, of the last three nucleotides of the guide RNA.
Embodiment A71 is the composition of any one of the preceding claims, wherein the guide RNA
is modified, and wherein the modification includes one or more of a phosphorothioate modification, a 2'-0Me modification, a 2'-0-MOE modification, a 2'-F
modification, a 2'-0-methine-4' bridge modification, a 3'-thiophosphonoacetate modification, or a 2'-deoxy modification.
Embodiment A72 is the composition of any one of the preceding claims, wherein the composition further comprises a pharmaceutically acceptable excipient.
Embodiment A73 is the composition of any one of the preceding claims, wherein the composition is associated with a lipid nanoparticle (LNP).

Embodiment A74 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector.
Embodiment A75 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, and wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector.
Embodiment A76 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, and wherein the viral vector is an adeno-associated virus (AAV) vector.
Embodiment A77 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10, AAVrh74, or AAV9 vector, wherein the number following AAV

indicates the AAV serotype.
Embodiment A78 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAV serotype 9 (AAV9) vector.
Embodiment A79 is the composition of claim 78, wherein the AAV serotype 9 vector is less than 5kb, less than 4.9kb, less than 4.85, less than 4.8, or less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs.
Embodiment A80 is the composition of claim 78 or 79, wherein the AAV serotype 9 vector is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs.
Embodiment A81 is the composition of any one of claims 78-80, wherein the AAV
serotype 9 vector is between 4.4-4.85 kb from ITR to ITR in size, inclusive of both ITRs.
Embodiment A82 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAVrhl 0 vector.
Embodiment A83 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAVrh74 vector.

Embodiment A84 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector comprises a tissue-specific promoter.
Embodiment A85 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector comprises a muscle-specific promoter, optionally wherein the muscle-specific promoter is a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, an SPc5-12 promoter, or a CK8e promoter.
Embodiment A86 is the composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector comprises any one or more of the following promoters: U6, H1, and 7SK promoter.
Embodiment A87 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein at least one guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 12, 15, 16, 20, 27, 28, 32, 33, and 35.
Embodiment A88 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ ID
NO: 711.
Embodiment A89 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ ID
NO: 711.
Embodiment A90 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 715-717.
Embodiment A91 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SluCas9, wherein at least one guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 131, 134, 135, 136 ,139, 144, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, and 225.
Embodiment A92 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 comprises the amino acid sequence of SEQ ID
NO: 712.
Embodiment A93 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 is a variant of the amino acid sequence of SEQ
ID NO: 712.

Embodiment A94 is the composition of any one of the preceding claims, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 718-720.
Embodiment A95 is the composition of claim 1, wherein the single nucleic acid molecule or the first nucleic acid comprises from 5' to 3' with respect to the plus strand:
the reverse complement of a nucleotide sequence encoding a first guide RNA scaffold sequence, the reverse complement of a nucleotide sequence encoding the first guide RNA
sequence, the reverse complement of a promoter for expression of the nucleotide sequence encoding the first guide RNA sequence, a promoter for expression of a nucleotide sequence encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9), a nucleotide sequence encoding the SaCas9 or SluCas9, a polyadenylation sequence, a promoter for expression of the second guide RNA in the same direction as the promoter for the SaCas9 or SluCas9, a nucleotide sequence encoding the second guide RNA
sequence, and a nucleotide sequence encoding the second guide RNA scaffold sequence.
Embodiment A96 is the composition of claim 95, wherein the promoter for expression of the nucleic acid encoding the first guide RNA sequence is a U6 promoter and the promoter for expression of the nucleic acid encoding the second guide RNA is a U6 promoter.
Embodiment A97 is the composition of claim 95 or 96, wherein the SaCas9 or SluCas9 comprise at least two nuclear localization signals (NLSs).
Embodiment A98 is the composition of claim 97, wherein the SaCas9 or SluCas9 comprise a c-Myc NLS fused to the N-terminus of the SaCas9 or SluCas9, optionally by means of a linker.
Embodiment A99 is the composition of claim 97 or 98, wherein the SaCas9 or SluCas9 comprise an 5V40 NLS fused to the C-terminus of the SaCas9 or SluCas9, optionally by means of a linker.
Embodiment A100 is the composition of any one of claims 97-99, wherein the SaCas9 or SluCas9 comprise a nucleoplasmin NLS fused to the C-terminus of the SaCas9 or SluCas9, optionally by means of a linker.
Embodiment A101 is the composition of any one of claims 97-100, wherein the SaCas9 or SluCas9 comprise:
i. a c-Myc NLS fused to the N-terminus of the SaCas9 or SluCas9, optionally by means of a linker, ii. an 5V40 NLS fused to the C-terminus of the SaCas9 or SluCas9, optionally by means of a linker, and iii. a nucleoplasmin NLS fused to the C-terminus of the 5V40 NLS, optionally by means of a linker, Embodiment A102 is the composition of any one of claims 95-101, wherein the scaffold sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901.
Embodiment A103 is the composition of any one of claims 95-101, wherein the scaffold sequence for the second guide RNA comprises the sequence of SEQ ID NO: 901.
Embodiment A104 is the composition of any one claims 95-103, wherein the single nucleic acid molecule or the first nucleic acid is less than 5kb, less than 4.9kb, 4.85, 4.8, or 4.75 kb in size.
Embodiment A105 is the composition of any one of claims 95-104, wherein the single nucleic acid molecule or the first nucleic acid is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb in size.
Embodiment A106 is the composition of claim 105, wherein the single nucleic acid molecule or the first nucleic acid is between 4.4-4.85 kb from ITR to ITR in size.
Embodiment A107 is a composition comprising a guide RNA encoded by a sequence comprising any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 or complements thereof Embodiment A108 is a composition comprising a guide RNA encoded by a sequence comprising any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 or complements thereof.
Embodiment A109 is the composition of any one of claims 1-108 for use in treating Duchenne Muscular Dystrophy (DMD).
Embodiment A110 is the composition of any one of claims 1-108 for use in making one or more double strand breaks in any one or more of exons 43, 44, 45, 50, 51, or 53 of the dystrophin gene.
Embodiment A111 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell the composition of any one of claims 1-107.
Embodiment A112 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i. a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:
1. a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, or 3000-3069;

2. a spacer sequence comprising at least 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, or 3000-3069; or 3. a spacer sequence that is at least 90% identical to any one of SEQ ID
NOs: 1-35, 1000-1078, 3000-3069; and ii. a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).
Embodiment A113 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
a. a nucleic acid molecule encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:
i. a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, or 4000-4251;
ii. a spacer sequence comprising at least 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, or 4000-4251; or iii. a spacer sequence that is at least 90% identical to any one of SEQ ID
NOs:
100-225, 2000-2116, or 4000-4251; and b. a nucleic acid molecule encoding Staphylococcus lugdunensis (SluCas9).
Embodiment A114 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i. a nucleic acid encoding a pair of guide RNAs comprising:
1. a first and second spacer sequence selected from any one of SEQ ID
NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15;
1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018;
2. a first and second spacer sequence comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of any one of SEQ ID NOs: 10 and 15;
and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16;
1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018;
or 3. a first and second spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005;
1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16;
1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and ii. a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).
Embodiment A115 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i. a nucleic acid encoding a pair of guide RNAs comprising:
1. a first and second spacer sequence selected from any one of SEQ ID
NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; and 146 and 148;
2. a first and second spacer sequence comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or 3. a first and second spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135;
131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131;
140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131;
145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150;

136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140;
151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145;
151 and 145; and 148 and 146; and a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9).
Embodiment A116 is the method of any one of the previous method or use claims, wherein the single nucleic acid molecule is delivered to the cell on a single vector.
Embodiment A117 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the spacer sequence is selected from any one of SEQ ID NOs: 12, 15, 16, 20, 27, 28, 32, 33, and 35.
Embodiment A118 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ ID NO: 711.
Embodiment A119 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ ID NO: 711.
Embodiment A120 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 715-717.
Embodiment A121 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SluCas9, wherein the spacer sequence is selected from any one of SEQ ID NOs: 131, 134, 135, 136 ,139, 144, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, and 225.
Embodiment A122 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SluCas9, wherein the SluCas9 comprises the amino acid sequence of SEQ ID NO: 712.
Embodiment A123 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SluCas9, wherein the SluCas9 is a variant of the amino acid sequence of SEQ ID NO: 712.
Embodiment A124 is the method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SluCas9, wherein the SluCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 718-720.

Embodiment A125 is a method of excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i. a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA
binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA; and ii. a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
wherein the pair of guide RNAs and SaCas9 excise a portion of the exon.
Embodiment A126 is a method of excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i. a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA
binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA; and ii. a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
wherein the pair of guide RNAs and SaCas9 excise a portion of the exon.
Embodiment A127 is the method of any one of the previous method claims, comprising a single nucleic acid molecule, wherein the single nucleic acid molecule is delivered to the cell on a single vector.
Embodiment A128 is the method of any one of the previous method claims, wherein a portion of a DMD gene is excised, and wherein the portions of the gene remaining after excision are rejoined with a one-nucleotide insertion.
Embodiment A129 is the method of any one of the previous method claims, wherein a portion of a DMD gene is excised, and wherein the portions of the exon remaining after excision are rejoined without a nucleotide insertion.
Embodiment A130 is the method of any one of the previous method claims, wherein a portion of a DMD gene is excised, wherein the size of the excised portion of the gene is between 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 75, 5 and 50, 5 and 25, 5 and 10, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 75, 20 and 50, 20 and 25, 50 and 250, 50 and 200, 50 and 150, 50 and 100, and 50 and 75 nucleotides.
Embodiment A131 is the method of any one of the previous method claims, wherein a portion of a DMD gene is excised, wherein the size of the excised portion of the exon is between 8 and 167 nucleotides.
Embodiment A132 is the method of any one of the previous method claims, wherein a portion of a DMD gene is excised, wherein the portion is within exon 43, 44, 45, 50, 51, or 53.
Embodiment A133 is the method of any one of the previous method claims, comprising a pair of guide RNAs, wherein the pair of guide RNA comprise a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;
1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16;
1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018.
Embodiment A134 is the method of any one of the previous method claims, comprising a pair of guide RNAs, wherein the pair of guide RNAs comprise a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135;
131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151;
141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140;
151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145;
and 148 and 146.
Embodiment A135 is the method of any one of the previous method claims, comprising SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ ID NO:
715.
Embodiment A136 is the composition or method of any one of the preceding claims, wherein the single nucleic acid molecule is an AAV vector, wherein the vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA
scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding a SaCas9, a polyadenylation sequence, a promoter for expression of a second sgRNA in the same direction as the promoter for SaCas9, a second sgRNA
guide sequence, and a second sgRNA scaffold sequence.

Embodiment A137 is the composition or method of claim 136, wherein the promoter for expression of the first sgRNA guide sequence is an hU6 promoter.
Embodiment A138 is the composition or method of any one of claims 136-137, wherein the promoter for expression of the second sgRNA guide sequence is an hU6 promoter.
Embodiment A139 is the composition or method of any one of claims 136-137, wherein the promoter for the expression of the first sgRNA guide sequence is an hU6 promoter, and the promoter for the expression of the second sgRNA guide sequence is an hU6 promoter.
Embodiment A140 is the composition or method of any one of claims 136-137, wherein the promoter for expression of the nucleic acid encoding the first sgRNA guide sequence is an 7SK promoter.
Embodiment A141 is the composition or method of any one of claims 136-137, wherein the promoter for expression of the nucleic acid encoding the second sgRNA guide sequence is an 7SK promoter.
Embodiment A142 is the composition or method of any one of claims 136-137, wherein the promoter for expression of the nucleic acid encoding the second sgRNA guide sequence is an Him promoter.
Embodiment A143 is the composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to a nucleic acid sequence encoding one or more nuclear localization sequences (NLSs).
Embodiment A144 is the composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS).
Embodiment A145 is the composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS).
Embodiment A146 is the composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS).
Embodiment A147 is the composition or method of any one of claims 143-146, wherein the one or more NLSs comprises an SV40 NLS.
Embodiment A148 is the composition or method of any one of claims 143-147, wherein the one or more NLSs comprises an c-Myc NLS.

Embodiment A149 is the composition or method of any one of claims 143-148, wherein the one or more NLSs comprises a nucleoplasmin NLS.
Embodiment A150 is the composition or method of any one of the preceding claims, wherein the nucleic acid encoding the guide RNA or the nucleic acid encoding the pair of guide RNAs comprises a sequence selected from any one of SEQ ID NOs: 600, 601, or 900-917, and wherein the composition or method comprises a nucleic acid encoding SluCas9.
Embodiment A151 is the composition or method of any one of the preceding claims, wherein the nucleic acid encoding the guide RNA or the nucleic acid encoding the pair of guide RNAs comprises a sequence selected from any one of SEQ ID NOs: 901-917, and wherein the composition or method comprises a nucleic acid encoding SluCas9.
Embodiment A152 is the composition of any one of the preceding claims, wherein the nucleic acid molecule encodes at least a first guide RNA and a second guide RNA.
Embodiment A153 is the composition of claim 152, wherein the nucleic acid molecule encodes a spacer sequence for the first guide RNA, a scaffold sequence for the first guide RNA, a spacer sequence for the second guide RNA, and a scaffold sequence for the second guide RNA.
Embodiment A154 is the composition of claim 152, wherein the spacer sequence for the first guide RNA and the spacer sequence for the second guide RNA are the same.
Embodiment A155 is the composition of claim 152, wherein the spacer sequence for the first guide RNA and the spacer sequence for the second guide RNA are different.
Embodiment A156 is the composition of any one of claims 153-155, wherein the scaffold sequence for the first guide RNA and the scaffold sequence for the second guide RNA are the same.
Embodiment A157 is the composition of any one of claims 153-154, wherein the scaffold sequence for the first guide RNA and the scaffold sequence for the second guide RNA are different.
Embodiment A158 is the composition of claim 157, wherein the scaffold sequence for the first guide RNA comprises a sequence selected from the group consisting of SEQ
ID NOs:
901-916, and wherein the scaffold sequence for the second guide RNA comprises a different sequence selected from the group consisting of SEQ ID NOs: 901-916.
DESCRIPTION OF FIGURES
[0009] Fig. 1 provides a simplified description of several representative vector configurations. Single-line arrows indicate directionality of expression of the sgRNA(s), while a double-line arrow indicates directionality of expression of the Cas9 protein.
For the promoter column, representative promoters for the expression of the sgRNAs are indicated. In a particular embodiment, the Cas9 promoter may be CK8e.

[0010] Fig. 2 shows an exemplary schematic of locus-specific indel profiling, as described in Example 2, including editing categories for insertions and deletions.

[0011] Figs. 3A-3B show the editing frequency and indel profile of selected SluCas9 and SaCas9 sgRNAs that target exon 45 of the DMD gene in human HEK293FT cells (Fig. 3A) and mouse Neuro-2a cells (Fig. 3B). A high-performing sgRNA for SpCas9 was included as reference (E45Sp52). Each bar represents a sgRNA and the bar height depicts the average total indel frequency.
Indel profiles are shown for each sgRNA as follows: solid black (1-nucleotide (nt) insertion leading to reframe, sometimes referred to as "RF.+1"); solid white (indels other than 1-nt insertion leading to reframe, sometimes referred to as "RF.Other"); checkerboard pattern (indels that disrupt the 6=nt window of the exon/intron boundaries, referred to as "Exon skipping");
diagonal lines (other indels, sometimes referred to as "OE"). Data are the average of 3 replicates.

[0012] Figs. 4A-4B show the editing frequency and indel profile of selected SluCas9 and SaCas9 sgRNAs that target exon 51 of the DMD gene in human HEK293FT cells (Fig. 4A) and mouse Neuro-2a cells (Fig. 4B). A high performing sgRNA for SpCas9 was included as reference (E51Sp32). Each bar represents a sgRNA and the bar height depicts the average total indel frequency.
Indel profiles are shown for each sgRNA as follows: solid black (1-nucleotide (nt) insertion leading to reframe, referred to as "RF.+1"); solid white (indels other than 1-nt insertion leading to reframe, referred to as "RF.Other"); checkerboard pattern (indels that disrupt the 6=nt window of the exon/intron boundaries, referred to as "Exon skipping"); diagonal lines (other indels, sometimes referred to as "OE" or "Other indel"). Data are the average of 3 replicates.

[0013] Figs. 5A-5B show the editing frequency and indel profile of selected SluCas9 and SaCas9 sgRNAs within exon 53 of the DMD gene in human HEK293FT cells (Fig. 5A) and mouse Neuro-2a cells (Fig. 5B). A high performing sgRNA for SpCas9 was included as reference (E53Sp63). Each bar represents a sgRNA and the bar height depicts the average total indel frequency.
Indel profiles are shown for each sgRNA using distinct colors as follows:
solid black (1-nucleotide (nt) insertion leading to reframe, referred to as "RF.+1"); solid white (indels other than 1-nt insertion leading to reframe, referred to as "RF.Other"); checkerboard pattern (indels that disrupt the 6=nt window of the exon/intron boundaries, referred to as "Exon skipping");
diagonal lines (other indels, referred to as "OE" or "Other indel"). Data are the average of 3 replicates.

[0014] Figs. 6A-D show editing frequency and indel profile of selected SaCas9-KKH and SluCas9 sgRNA pairs within exon 45 in HEK293FT cells (Fig. 6B) and Neuro-2a cells (Fig. 6D). Fig.
6A shows a schematic of editing. A single cut sgRNA for SaCas9 (SaCas9-4) was included as reference (Fig. 6C). Each bar represents a sgRNA and the bar height depicts the average total indel frequency. Indel profiles are shown for each sgRNA using distinct patterns.
Error bar = upper limit of SEM for each indel group.

[0015] Fig. 7A
shows the nucleotide composition and RNA secondary structure of the stem-loop I in different SluCas9 single-guide RNA scaffolds. Key differences in the sequence and secondary structure between Slu-VCGT-4.5, Slu-VCGT-4 and Slu-VCGT-5 are depicted. Squares and triangles show the difference in the secondary structure in the upper stem.
Diamond and pentagon shapes show the single nucleotide change in the bottom stem.

[0016] Fig. 7B
is a histogram showing the percentage of different types of indels generated by two SluCas9 sgRNA candidates: SluCas9-23 and SluCas9-24. For each guide RNA, three scaffolds were tested, including Slu-VCGT-4.5, Slu-VCGT-4 and Slu-VCGT-5. Each sgRNA was tested at three different RNP doses. The exact amounts of SluCas9 protein and sgRNA
tested were: 6.75 pmo1:37.5pmo1 for low dose, 12.5pmo1:75pmo1 for middle dose, and 25pmo1:150pmol for high dose.
The different colors of the bars in the histogram represent different types of indels generated by sgRNAs. Black represents the percentage of +1 bp insertions. White represents the percentage of other insertions and deletions that have the potential to restore the reading frame of particular DMD
patient mutations of interest. These are referred to as "RF other", which represents the sum of 2, 5, 8, 11 bp deletions within the alignment window of -20bp to +20bp around the Cas9 cut site. The remaining indels shown in pattern are classified as "Other indels".

[0017] Fig. 8 shows editing frequency and indel profile of selected SluCas9 sgRNA pairs within exon 45 in primary human skeletal muscle myoblasts (HsMMs). Two single cut sgRNAs for SpCas9 (EX-145, E45Sp52) and SluCas9 (SluCas9-24) were included as reference.
Each bar represents a sgRNA or a sgRNA pair and the bar height depicts the average total indel frequency.
Indel profiles are shown using distinct patterns. Error bar = upper limit of SD for each indel group.

[0018] Figs. 9A
and 9B show editing frequency and indel profile of selected SaCas9-KKH
and SluCas9 sgRNA pairs within exon 51 in HEK293FT cells. Each bar represents a sgRNA or a sgRNA pair and the bar height depicts the average total indel frequency. Indel profiles are shown using distinct patterns. Error bar = upper limit of SD for each indel group.

[0019] Figs.
10A and 10B show editing frequency and indel profile of selected AAV vectors in C2C12 myotubes. Three test guides are shown for Fig. 10A and 4 test guides for Fig. 10B. Each bar represents a different AAV configuration and the bar height depicts the average total indel frequency.
Indel profiles are shown using distinct patterns. Error bar = upper limit of SD for each indel group.

[0020] Fig. 11 shows vector genome quantitation of selected AAV vectors in C2C12 myotubes. Each bar represents a different AAV configuration and the bar height depicts the average vector genome copy number per lag DNA. Error bar = upper limit of SD.

[0021] Figs.
12A and 12B show transgene expression of selected AAV vectors in C2C12 myotubes. Fig. 12A: each bar represents a different AAV configuration and the bar height depicts the average Cas9 transgene copy per ug RNA. Cas9 nucleases are shown using distinct patterns. Fig.

12B: each bar represents a different sgRNA and the bar height depicts the average sgRNA transgene copy per ug RNA. Promoter combinations are shown using distinct patterns.
Error bar = upper limit of SD.

[0022] Fig. 13 shows Cas9 localization of selected AAV vectors in C2C12 myotubes. In Fig.
13 and throughout the application, single-line arrows indicate directionality of expression of the sgRNA(s), while a double-line or "thick" arrow (with or without an embedded "X") indicates directionality of expression of the Cas9 protein. SluCas9's location is shown in the bottom panel of each; MYOG is shown with representative arrows, DAPI is shown with representative bold, larger arrows. Image shown at 20-25 gm scale.

[0023] Figs.
14A and 14B show editing frequency and indel profile of selected AAV vectors in heart (FIG. 14A) and quadriceps (FIG. 14B) of dEx44 mouse model. Each bar represents a different AAV configuration and the bar height depicts the average total indel frequency. Indel profiles are shown using distinct patterns. Error bar = upper limit of SD for each indel group.

[0024] Figs.
15A and 15B show dystrophin restoration of selected AAV vectors in heart (FIG. 15A) and quadriceps (FIG. 15B) of dEx44 mouse model. Each group represents a different AAV configuration and the horizontal bar depicts the average total dystrophin protein level.

[0025] Figs.
16A and 16B show vector genome quantitation of selected AAV vectors in heart (FIG. 16A) and quadriceps (FIG. 16B) of dEx44 mouse model. Each group represents a different AAV configuration and the horizontal bar depicts the average vector genome copy number per ug DNA.

[0026] Figs.
17A and 17B show Cas9 and sgRNA transgene expression of selected AAV
vectors in heart (FIG. 17A) and quadriceps (FIG. 17B) of dEx44 mouse model.
Each group represents a different AAV configuration and the horizontal bar depicts the average transgene copy number per ug RNA. Closed circles indicate Cas9 mRNA expression and open circles indicate sgRNA expression.

[0027] Figs.
18A-18C show editing frequency and indel profile of selected Cas12i2 guide RNAs used with Cas12i2 endonuclease in exon 45 in HEK293FT cells. FIG. 18A
shows a schematic of the experiment, FIG. 18B shows indel frequency for selected pairs of guide RNAs, and FIG. 18C
shows a single cut sgRNA for SaCas9 (SaCas9-4) and for SluCas9 (SluCas9-24) for reference.
DETAILED DESCRIPTION

[0028]
Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims and included embodiments.

[0029] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a guide" includes a plurality of guides and reference to "a cell" includes a plurality of cells and the like.

[0030] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of "comprise", "comprises", "comprising", contain", "contains", "containing", "include", "includes", and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0031] Unless specifically noted in the specification, embodiments in the specification that recite "comprising" various components are also contemplated as "consisting of' or "consisting essentially of' the recited components; embodiments in the specification that recite "consisting of' various components are also contemplated as "comprising" or "consisting essentially of' the recited components; and embodiments in the specification that recite "consisting essentially of' various components are also contemplated as "consisting of' or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims).
The term "or" is used in an inclusive sense, i.e., equivalent to "and/or," unless the context clearly indicates otherwise.

[0032] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
I. Definitions

[0033] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

[0034]
"Polynucleotide," "nucleic acid," and "nucleic acid molecule," are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A
nucleic acid "backbone" can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds ("peptide nucleic acids" or PNA; PCT No.
WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2' methoxy or 2' halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, 06-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and 04-alkyl-pyrimidines; US Pat. No. 5,378,825 and PCT No. WO
93/13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids can include one or more "abasic" residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No. 5,585,481). A
nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs).
Nucleic acid includes "locked nucleic acid" (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

[0035] "Guide RNA", "guide RNA", and simply "guide" are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA
(also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "guide RNA" refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences. For clarity, the terms "guide RNA" or "guide" as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. In general, in the case of a DNA nucleic acid construct encoding a guide RNA, the U residues in any of the RNA sequences described herein may be replaced with T
residues, and in the case of a guide RNA construct encoded by any of the DNA sequences described herein, the T residues may be replaced with U residues.

[0036] As used herein, a "spacer sequence," sometimes also referred to herein and in the literature as a "spacer," "protospacer," "guide sequence," or "targeting sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA
to a target sequence for cleavage by a Cas9. A guide sequence can be 24, 23, 22, 21, 20 or fewer base pairs in length, e.g., in the case of Staphylococcus lugdunensis (i.e., SluCas9) or Staphylococcus aureus i.e.,( SaCas9) and related Cas9 homologs/orthologs. In preferred embodiments, a guide/spacer sequence in the case of SluCas9 or SaCas9 is at least 20 base pairs in length, or more specifically, within 20-25 base pairs in length (see, e.g., Schmidt et al., 2021, Nature Communications, "Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases").
Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. For example, in some embodiments, the guide sequence comprises at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from SEQ ID
NOs: 1-35 (for SaCas9), and 100-225 (for SluCas9). In some embodiments, the guide sequence comprises a sequence selected from SEQ ID NOs: 1-35, 100-225, 1000-1078, 2000-2116, 3000-3069, or 4000-4251. In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, in some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-35, 100-225, 1000-1078, 2000-2116, 3000-3069, or 4000-4251. In some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-35, 100-225, 1000-1078, 2000-2116, 3000-3069, or 4000-4251. In some embodiments, the guide sequence and the target region may be 100%
complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides. In some embodiments, the guide sequence and the target region do not contain any mismatches.

[0037] In some embodiments, the guide sequence comprises a sequence selected from SEQ
ID NOs: 1-35, 100-225, 1000-1078, 2000-2116, 3000-3069, or 4000-4251, wherein if the 5' terminal nucleotide is not guanine, one or more guanine (g) is added to the sequence at its 5' end. The 5' g or gg is required in some instances for transcription, for example, for expression by the RNA polymerase III-dependent U6 promoter or the T7 promoter. In some embodiments, a 5' guanine is added to any one of the guide sequences or pairs of guide sequences disclosed herein.

[0038] Target sequences for Cas9s include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for a Cas9 is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be complementary to a target sequence", it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0039] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA
together with a Cas9. In some embodiments, the guide RNA guides the Cas9 such as Cas9 to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence, which can be followed by cleaving or nicking (in the context of a modified "nickase"
Cas9).

[0040] As used herein, a first sequence is considered to "comprise a sequence with at least X% identity to" a second sequence if an alignment of the first sequence to the second sequence shows that X% or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100%
identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA
(generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.

[0041] "mRNA"
is used herein to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2'-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxy ribose residues, or a combination thereof.

[0042] Guide sequences useful in the guide RNA compositions and methods described herein are shown, for example, in Table 1A, Table 1B, and Table 5 and throughout the specification.

[0043] As used herein, a "target sequence" refers to a sequence of nucleic acid in a target gene that has complementarity to at least a portion of the guide sequence of the guide RNA. The interaction of the target sequence and the guide sequence directs a Cas9 to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence.

[0044] As used herein, "treatment" refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease or development of the disease (which may occur before or after the disease is formally diagnosed, e.g., in cases where a subject has a genotype that has the potential or is likely to result in development of the disease), arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of DMD
may comprise alleviating symptoms of DMD.

[0045] As used herein, "ameliorating" refers to any beneficial effect on a phenotype or symptom, such as reducing its severity, slowing or delaying its development, arresting its development, or partially or completely reversing or eliminating it. In the case of quantitative phenotypes such as expression levels, ameliorating encompasses changing the expression level so that it is closer to the expression level seen in healthy or unaffected cells or individuals.

[0046] A
"pharmaceutically acceptable excipient" refers to an agent that is included in a pharmaceutical formulation that is not the active ingredient. Pharmaceutically acceptable excipients may e.g., aid in drug delivery or support or enhance stability or bioavailability.

[0047] The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined.

[0048] As used herein, "Staphylococcus aureus Cas9" may also be referred to as SaCas9, and includes wild type SaCas9 (e.g., SEQ ID NO: 711) and variants thereof A
variant of SaCas9 comprises one or more amino acid changes as compared to SEQ ID NO: 711, including insertion, deletion, or substitution of one or more amino acids, or a chemical modification to one or more amino acids.

[0049] As used herein, "Staphylococcus lugdunensis Cas9" may also be referred to as SluCas9, and includes wild type SluCas9 (e.g., SEQ ID NO: 712) and variants thereof A variant of SluCas9 comprises one or more amino acid changes as compared to SEQ ID NO:
712, including insertion, deletion, or substitution of one or more amino acids, or a chemical modification to one or more amino acids.

Compositions

[0050] Provided herein are compositions useful for treating Duchenne Muscular Dystrophy (DMD), e.g., using a single or multiple (e.g., at least 2) nucleic acid molecule encoding 1) one or more guide RNAs comprising one or more guide sequences of Table 1A, Table 1B, or Table 5; and 2) SaCas9 (for SEQ ID NOs: 1-35, 1000-1078 or 3000-3069) or SluCas9 (for SEQ
ID NOs: 100-225, 2000-2116, or 4000-4251). Such compositions may be administered to subjects having or suspected of having DMD.

[0051] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of a single nucleic acid molecule comprising: i) a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; or ii) a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii) a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and one to three guide RNAs.

[0052] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of at least two nucleic acid molecules comprising a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9);
and a second nucleic acid that does not encode a SaCas9 or SluCas9 and encodes any one of i) at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or ii) from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii) from one to six guide RNAs.

[0053] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of at least two nucleic acid molecules comprising a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and i) at least one, at least two, or at least three guide RNAs; or ii) from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid;
or iii) one to three guide RNAs; and a second nucleic acid that does not encode a SaCas9 or SluCas9, optionally wherein the second nucleic acid comprises any one of i) at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or ii) from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii) from one to six guide RNAs.

[0054] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of at least two nucleic acid molecules comprising a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SaCas9 or SluCas9 and encodes from one to six guide RNAs.

[0055] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of at least two nucleic acid molecules comprising a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and at least two guide RNAs, wherein at least one guide RNA binds upstream of sequence to be excised and at least one guide RNA binds downstream of sequence to be excised; and a second nucleic acid that does not encode a SaCas9 or SluCas9 and encodes at least one additional copy of the guide RNAs encoded in the first nucleic acid. In some embodiments, the guide RNA excises a portion of a DMD gene, optionally an exon, intron, or exon/intron junction.

[0056] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of at least two nucleic acid molecules comprising a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and a first and a second guide RNA that function to excise a portion of a DMD gene; and a second nucleic acid encoding at least 2 or at least 3 copies of the first guide RNA and at least 2 or at least 3 copies of the second guide RNA.

[0057] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of one or more nucleic acid molecules encoding an endonuclease and a pair of guide RNAs, wherein each guide RNA targets a different sequence in a DMD gene, wherein the endonuclease and pair of guide RNAs are capable of excising a target sequence in DNA that is between 5-250 nucleotides in length. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease is SpCas9, SaCas9, or SluCas9. In some embodiments, the endonuclease is not a class 2, type V Cas endonuclease. In some embodiments, the excised target sequence comprises a splice acceptor site or a splice donor site. In some embodiments, the excised target sequence comprises a premature stop codon in the DMD gene.
In some embodiments, the excised target sequence does not comprise an entire exon of the DMD
gene. In some embodiments, any of the methods and/or ribonucleoprotein complexes disclosed herein do not destroy/specifically alter the sequence of a splice acceptor site, splice donor site, or premature stop codon site.

[0058] In some embodiments, the guide RNA in the composition comprises any one of the following:
a. when SaCas9 is used, one or more spacer sequences selected from any one of SEQ ID
NOs: 1-35, 1000-1078, and 3000-3069; or b. when SluCas9a is used, one or more spacer sequences selected from any one of SEQ
ID NOs: 100-225, 2000-2116, and 4000-4251; or c. when SaCas9 is used, one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID

NOs: 1-35, 1000-1078, and 3000-3069; or d. when SluCas9a is used, one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ
ID
NOs: 100-225, 2000-2116, and 4000-4251; or e. when SaCas9 is used, one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; or f. when SluCas9a is used, one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; or g. when SaCas9 is used with at least two guide RNAs, a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;

1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10;
1005 and 1017; 16 and 1017; and 16 and 1018; or h. when SaCas9 is used with at least two guide RNAs, at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12;
1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; or i. when SaCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001;
16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12;
1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; or j. when SluCas9 is used with at least two guide RNAs, a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or k. when SluCas9 is used with at least two guide RNAs, at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136;
139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149;

and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or 1. when SluCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134;

and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or m. when SluCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150; and v. SEQ ID Nos: 146 and 148; or n. when SaCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016.

[0059] In some embodiments, the disclosure provides for a composition comprising: a) one or more nucleic acid molecules encoding a SluCas9 and b) a first spacer sequence and a second spacer sequence selected from SEQ ID Nos: 148 and 134. In some embodiments, the composition comprises: a) one or more nucleic acid molecules encoding a SluCas9 and b) a first spacer sequence and a second spacer sequence selected from SEQ ID Nos: 145 and 131. In some embodiments, the composition comprises: a) one or more nucleic acid molecules encoding a SluCas9 and b) a first spacer sequence and a second spacer sequence selected from SEQ ID Nos: 144 and 149. In some embodiments, the composition comprises: a) one or more nucleic acid molecules encoding a SluCas9 and b) a first spacer sequence and a second spacer sequence selected from SEQ
ID Nos: 144 and 150.
In some embodiments, the composition comprises: a) one or more nucleic acid molecules encoding a SluCas9 and b) a first spacer sequence and a second spacer sequence selected from SEQ ID Nos: 146 and 148. In some embodiments, the composition comprises: a) one or more nucleic acid molecules encoding a SaCas9 (e.g., an SaCas9-KKH) and b) a first spacer sequence and a second spacer sequence selected from SEQ ID Nos: 12 and 1013. In some embodiments, the composition comprises: a) one or more nucleic acid molecules encoding a SaCas9 (e.g., an SaCas9-KKH) and b) a first spacer sequence and a second spacer sequence selected from SEQ ID Nos:
12 and 1016.

[0060] In some embodiments, the one or more guide RNAs direct the Cas9 to a site in or near any one of exon 43, 44, 45, 50, 51, or 53 of dystrophin. For example, the Cas9 may be directed to cut within 10, 20, 30, 40, or 50 nucleotides of a target sequence.

[0061] The disclosure herein may reference a "first and a second spacer" or a "first and a second guide RNA, gRNA, or sgRNA" followed by one or more pairs of specific sequences. It should be noted that the order of the sequences in the pair is not intended to be restricted to the order in which they are presented. For example, the phrase "the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 15" could mean that the first sgRNA comprises the sequence of SEQ ID NO: 10 and the second sgRNA sequence comprises the sequence of SEQ ID
NO: 15, or this phrase could mean that the first sgRNA comprises the sequence of SEQ ID NO: 15 and the second sgRNA sequence comprises the sequence of SEQ ID NO: 10.

[0062] In some embodiments, a composition comprising a single nucleic acid molecule encoding one or more guide RNAs and a Cas9 is provided, wherein the single nucleic acid molecule comprises:
a. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
b. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
c. a first nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
d. a first nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
e. a first nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
f. a first nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
g. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016;
1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12;
1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16;
1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
h. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;
1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12;
1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018;
and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
i. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of SEQ
ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10;
1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
j. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134;
149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150;
136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
k. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151;
146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
1. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of SEQ
ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136;
139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139;
131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
m. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150;
v. SEQ ID Nos: 146 and 148; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or n. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).

[0063] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of one or more nucleic acid molecules encoding a Staphylococcus lugdunensis (SluCas9) and at least two guide RNAs, wherein a first and second guide RNA target different sequences in a DMD gene, wherein the first and a second guide RNA comprise a sequence that is at least 90%
identical to a first and second spacer sequence selected from any one of:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150;
v. SEQ ID Nos: 146 and 148.

[0064] In some embodiments, a composition is provided comprising, consisting of, or consisting essentially of one or more nucleic acid molecules encoding an endonuclease and at least two guide RNAs, wherein the guide RNAs each target a different sequence in a DMD gene, wherein the guide RNAs each comprise a sequence that is at least 90% identical to a first and second spacer sequence selected from any one of:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).

[0065] In some embodiments, the first and/or the second nucleic acid, if present, comprises at least two guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least three guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least four guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least five guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least six guide RNAs. In some embodiments, the first nucleic acid comprises an endonuclease and at least one, at least two, or at least three guide RNAs. In some embodiments, the first nucleic acid comprises an endonuclease and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid. In some embodiments, the first nucleic acid comprises an endonuclease and from one to three guide RNAs.

[0066] In some embodiments, the second nucleic acid, if present, encodes at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs.
In some embodiments, the second nucleic acid, if present, encodes from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid In some embodiments, the second nucleic acid, if present, encodes from one to six guide RNAs. In some embodiments, the second nucleic acid, if present, encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 guide RNAs. In some embodiments, the second nucleic acid, if present, encodes 2, 3, 4, 5, or 6 guide RNAs.

[0067] In some embodiments comprising at least two nucleic acid molecules, the guide RNA
encoded by the first nucleic acid and the second nucleic acid are the same. In some embodiments comprising at least two nucleic acid molecules, the guide RNA encoded by the first nucleic acid and the second nucleic acid are different. In some embodiments comprising at least two nucleic acid molecules, at least one guide RNA binds to a target sequence within an exon in the DMD gene that is upstream of a premature stop codon, and wherein at least one guide RNA binds to a target sequence within an exon in the DMD gene that is downstream of a premature stop codon.
In some embodiments comprising at least two nucleic acid molecules, the same guide RNA is encoded by the nucleic acid of the first and second nucleic acid molecule. In some embodiments comprising at least two nucleic acid molecules, the second nucleic acid molecule encodes a guide RNA that binds to the same target sequence as the guide RNA in the first nucleic acid molecule. In some embodiments comprising at least two nucleic acid molecules, the second nucleic acid molecule encodes at least 2, at least 3, at least 4, at least 5, or at least 6 guide RNAs, wherein the guide RNAs in the second nucleic acid molecule bind to the same target sequence as the guide RNA in the first nucleic acid molecule.

[0068] In some embodiments, the guide RNA binds to one or more target sequences within a DMD gene. In some embodiments, the guide RNA binds to one or more target sequences within an exon of the DMD gene. In some embodiments comprising two guide RNAs, i) each guide RNA
targets a sequence within an exon; ii) one guide RNA targets a sequence within an exon and one targets a sequence within an intron; or iii) each guide RNA targets a sequence within an intron.

[0069] In some embodiments comprising at least two guide RNAs, i) each guide RNA targets the same genomic target sequence; ii) each guide RNA targets a different target sequence; or iii) at least one guide RNA targets one sequence and at least one guide RNA targets a different sequence.

[0070] In some embodiments comprising a guide RNA that binds to an exon of the DMD gene, the exon is selected from exon 43, 44, 45, 50, 51, and 53.

[0071] In some embodiments comprising at least two guide RNAs that binds to an exon of the DMD gene, at least one guide RNA binds to a target sequence within an exon in the DMD gene, and at least one guide RNA binds to a different target sequence within the same exon in the DMD gene.

[0072] In some embodiments comprising at least two guide RNAs, at least one guide RNA binds to a target sequence within an exon that is upstream of a premature stop codon, and at least one guide RNA binds to a target sequence within an exon that is downstream of a premature stop codon.

[0073] In some embodiments comprising at least two guide RNAs, at least one guide RNA binds to a target sequence within an exon in the DMD gene, and at least one guide RNA binds to a different target sequence within the same exon in the DMD gene, wherein when expressed in vivo or in vivo, a portion of the exon is excised. In some embodiments comprising at least two guide RNAs, wherein once expressed in vivo or in vivo, the guide RNAs excise a portion of the exon.

[0074] In some embodiments comprising at least two guide RNAs, wherein once expressed in vivo or in vivo, the guide RNAs excise a portion of the exon, and wherein the portion of the exon remaining after excision are rejoined with a one nucleotide insertion. In some embodiments comprising at least two guide RNAs, wherein once expressed in vivo or in vivo, the guide RNAs excise a portion of the exon, wherein the portion of the exon remaining after excision is rejoined without a nucleotide insertion.

[0075] In some embodiments comprising at least two guide RNAs, wherein once expressed in vivo or in vivo, the guide RNAs excise a portion of the exon, wherein the size of excised portion of the exon is between 5, 6, 7, 8, 9, 10, 15, or 20 and 250 nucleotides in length. In some embodiments comprising at least two guide RNAs, once expressed in vivo or in vivo, the guide RNAs excise a portion of the exon, wherein the size of excised portion of the exon is between 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 75, 5 and 50, 5 and 25, 5 and 10, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 75, 20 and 50, 20 and 25, 50 and 250, 50 and 200, 50 and 150, 50 and 100, and 50 and 75 nucleotides.

[0076] In some embodiments comprising at least two guide RNAs, wherein once expressed in vivo or in vivo, the guide RNAs excise a portion of the exon, wherein the size of excised portion of the exon is between 8 and 167 nucleotides.

[0077] In some embodiments, a guide RNA and a Cas9 are provided on a single nucleic acid molecule. In some embodiments, the single nucleic acid molecule comprises a nucleic acid encoding a guide RNA and a nucleic acid encoding a SaCas9 or SluCas9. In some embodiments, two guide RNAs and a Cas9 are provided on a single nucleic acid molecule. In some embodiments, the single nucleic acid molecule comprises a nucleic acid encoding a first guide RNA, a nucleic acid encoding a second guide RNA, and a nucleic acid encoding a SaCas9 or SluCas9. In some embodiments, the spacer sequences of the first and second guide RNAs are identical. In some embodiments, the spacer sequences of the first and second guide RNAs are not identical.

[0078] In some embodiments, the single nucleic acid molecule is a single vector. In some embodiments, the single vector expresses the one or two guide RNAs and Cas9 according to the schemes of Figure 1. In some embodiments, a guide RNA and a Cas9 are provided on a single vector.
In some embodiments, the single vector comprises a nucleic acid encoding a guide RNA and a nucleic acid encoding a SaCas9 or SluCas9. In some embodiments, two guide RNAs and a Cas9 are provided on a single vector. In some embodiments, the single vector comprises a nucleic acid encoding a first guide RNA, a nucleic acid encoding a second guide RNA, and a nucleic acid encoding a SaCas9 or SluCas9. In some embodiments, the spacer sequences of the first and second guide RNAs are identical. In some embodiments, the spacer sequences of the first and second guide RNAs are not identical.

[0079] Each of the guide sequences shown in Table 1A, Table 1B, and Table 5 may further comprise additional nucleotides to form or encode a crRNA, e.g., using any known sequence appropriate for the Cas9 being used. In some embodiments, the crRNA comprises (5' to 3') at least a spacer sequence and a first complementarity domain. The first complementary domain is sufficiently complementary to a second complementarity domain, which may be part of the same molecule in the case of an sgRNA or in a tracrRNA in the case of a dual or modular gRNA, to form a duplex. See, e.g., US 2017/0007679 for detailed discussion of crRNA and gRNA domains, including first and second complementarity domains.

[0080] A single-molecule guide RNA (sgRNA) can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and/or an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins.
In particular embodiments, the disclosure provides for an sgRNA comprising a spacer sequence and a tracrRNA
sequence.

[0081] An exemplary scaffold sequence suitable for use with SaCas9 to follow the guide sequence at its 3' end is:
GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAATGCCGT
GTTTATCTCGTCAACTTGTTGGCGAGA (SEQ ID NO: 500) in 5' to 3' orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3' end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 500, or a sequence that differs from SEQ
ID NO: 500 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0082] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3' end is referred to as "SaScaffoldV1" and is:
GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGT
CAACTTGTTGGCGAGAT (SEQ ID NO: 501) in 5' to 3' orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3' end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 910, or a sequence that differs from SEQ ID NO: 910 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0083] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3' end is referred to as "SaScaffoldV2" and is:
GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAATGCCGT
GTTTATCTCGTCAACTTGTTGGCGAGAT (SEQ ID NO: 502) in 5' to 3' orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3' end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 911, or a sequence that differs from SEQ
ID NO: 911 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0084] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3' end is referred to as "SaScaffoldV3" and is:
GTTTAAGTACTCTGGAAACAGAATCTACTTAAACAAGGCAAAATGCCGTGTTTATCTCGT
CAACTTGTTGGCGAGAT (SEQ ID NO: 503) in 5' to 3' orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3' end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 912, or a sequence that differs from SEQ ID NO: 912 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0085] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3' end is referred to as "SaScaffoldV5" and is:
GTTTCAGTACTCTGGAAACAGAATCTACTGAAACAAGGCAAAATGCCGTGTTTATCTCGT
CAACTTGTTGGCGAGAT (SEQ ID NO: 932) in 5' to 3' orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3' end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%

identical to SEQ ID NO: 932, or a sequence that differs from SEQ ID NO: 932 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0086] Two exemplary scaffold sequences suitable for use with SluCas9 to follow the guide sequence at its 3' end is:
GTTTTAGTACTCTGGAAACAGAATCTACTGAAACAAGACAATATGTCGTGTTTATCCCAT
CAATTTATTGGTGGGA (SEQ ID NO: 900), and GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTGAAACAAGACAATATGTCGT
GTTTATCCCATCAATTTATTGGTGGGA (SEQ ID NO: 601) in 5' to 3' orientation. In some embodiments, an exemplary sequence for use with SluCas9 to follow the 3' end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 900 or SEQ ID NO: 601, or a sequence that differs from SEQ ID NO:
900 or SEQ ID NO: 601 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0087] Exemplary scaffold sequences suitable for use with SluCas9 to follow the guide sequence at its 3' end are also shown below in the 5' to 3' orientation:
Scaffold SEQ Scaffold Sequence (5' to 3') Homology Streak of ID ID NO to Slu v5 Homology to Slu v5 (# nucleotides) Wildtype 900 GTTTTAGTACTCTGGAAACAGAATCTACTGAA N/A N/A
ACAAGACAATATGTCGTGTTTATCCCATCAAT
TTATTGGTGGGAT
Slu- 601 GTTTAAGTACTCTGTGCTGGAAACAGCACAG N/A N/A
VCGT- AATCTACTGAAACAAGACAATATGTCGTGTTT
4.5 ATCCCATCAATTTATTGGTGGGA
Slu v5 901 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 100.00% 77 ACAAGACAATATGTCGTGTTTATCCCATCAAT
TTATTGGTGGGAT
Slu v5-1 902 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 87.50% 47 AGACAATATGTCGTGTTTATCCCATCAATTTA
TTGGTGGGAT
Slu v5-2 903 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 96.10% 37 ACAAGgCAAaATGcCGTGTTTATCCCATCAATT
TATTGGTGGGAT
Slu v5-3 904 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 94.81% 48 ACAAGACAATATGTCGcgcccaTCCCATCAATTT
ATTGGTGGGAT
Slu v5-4 905 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 91.55% 55 ACAAGACAATATGTCGTGTTTATgggTTgAATT
TATTcGacccAT
Slu v5-5 906 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 83.75% 31 AGgCAAaATGcCGTGTTTATCCCATCAATTTAT
TGGTGGGAT
Slu v5-6 907 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 82.50% 23 AGACAATATGTCGcgcccaTCCCATCAATTTATT
GGTGGGAT
Slu v5-7 908 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 78.38% 25 AGACAATATGTCGTGTTTATgggTTgAATTTAT

TcGacccAT
Slu v5-8 909 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 90.91% 37 ACAAGgCAAaATGcCGcgcccaTCCCATCAATTTA
TTGGTGGGAT
Slu v5-9 910 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 87.32% 37 ACAAGgCAAaATGcCGTGTTTATgggTTgAATTT
ATTcGacccAT
Slu v5- 911 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 82.89% 48 ACAAGACAATATGTCGcgcccaTgggTTgAATTTA
TTcGacccAT
Slu v5- 912 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 78.75% 23 11 AGgCAAaATGcCGcgcccaTCCCATCAATTTATTG
GTGGGAT
Slu v5- 913 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 74.32% 9 12 AGgCAAaATGcCGTGTTTATgggTTgAATTTATTc GacccAT
Slu v5- 914 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 70.89% 18 13 AGACAATATGTCGcgcccaTgggTTgAATTTATTc GacccAT
Slu v5- 915 GTTTCAGTACTCTGGAAACAGAATCTACTGAA 78.95% 37 14 ACAAGgCAAaATGcCGcgcccaTgggTTgAATTTAT
TcGacccAT
Slu v5- 916 GTTTggTaACcTaGGAAACTagATCTTaccAAACA 67.09% 8 AGgCAAaATGcCGcgcccaTgggTTgAATTTATTcGa cccAT
Slu v4 917 GTTTCAGTACTCTGTGCTGGAAACAGCACAGA N/A N/A
ATCTACTGAAACAAGACAATATGTCGTGTTTA
TCCCATCAATTTATTGGTGGGAT

[0088] In some embodiments, the scaffold sequence suitable for use with SluCas9 to follow the guide sequence at its 3' end is selected from any one of SEQ ID NOs: 900 or 601, or 901-917 in 5' to 3 orientation (see below). In some embodiments, an exemplary sequence for use with SluCas9 to follow the 3' end of the guide sequence is a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one off SEQ ID
NOs: 900 or 601, or 901-917, or a sequence that differs from any one of SEQ ID
NOs: 900 or 601, or 901-917 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0089] In some embodiments, the scaffold sequence suitable for use with SluCas9 to follow the guide sequence at its 3' end is selected from any one of SEQ ID NOs: 901-917 in 5' to 3 orientation (see below). In some embodiments, an exemplary sequence for use with SluCas9 to follow the 3' end of the guide sequence is a sequence that is at least 65%, 70%, 75%, 80%, 85%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one off SEQ ID NOs: 901-917, or a sequence that differs from any one of SEQ ID NOs: 901-917 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.
[0090] In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 900. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 601. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 900. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 901. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 902. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 903. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 904. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID
NO: 905. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 906. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 907. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID
NO: 908. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 909. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID
NO: 910. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 911. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 912. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID
NO: 913. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 914. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID
NO: 915. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 916. In some embodiments, the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence comprising SEQ ID NO: 917. In some embodiments, comprising a pair of gRNAs, one of the gRNAs comprises a sequence selected from any one of SEQ ID NOs: 900 or 601, or 901-917. In some embodiments, comprising a pair of gRNAs, both of the gRNAs comprise a sequence selected from any one of SEQ ID NOs: 900 or 601, or 901-917. In some embodiments, comprising a pair of gRNAs, the nucleotides 3' of the guide sequence of the gRNAs are the same sequence.
In some embodiments, comprising a pair of gRNAs, the nucleotides 3' of the guide sequence of the gRNAs are different sequences.

[0091] In some embodiments, the scaffold sequence comprises one or more alterations in the stem loop 1 as compared to the stem loop 1 of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the stem loop 2 as compared to the stem loop 2 of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID
NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901).
In some embodiments, the scaffold sequence comprises one or more alterations in the tetraloop as compared to the tetraloop of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the repeat region as compared to the repeat region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO:
901). In some embodiments, the scaffold sequence comprises one or more alterations in the anti-repeat region as compared to the anti-repeat region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the linker region as compared to the linker region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID
NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901).
See, e.g., Nishimasu et al., 2015, Cell, 162:1113-1126 for description of regions of a scaffold.

[0092] Where a tracrRNA is used, in some embodiments, it comprises (5' to 3') a second complementary domain and a proximal domain. In the case of a sgRNA, guide sequences together with additional nucleotides (e.g., SEQ ID Nos: 500, or 910-912 (for SaCas9), and 900 or 601, or 901-917 (for SluCas9)) form or encode a sgRNA. In some embodiments, an sgRNA
comprises (5' to 3') at least a spacer sequence, a first complementary domain, a linking domain, a second complementary domain, and a proximal domain. A sgRNA or tracrRNA may further comprise a tail domain. The linking domain may be hairpin-forming. See, e.g., US 2017/0007679 for detailed discussion and examples of crRNA and gRNA domains, including second complementarity domains, linking domains, proximal domains, and tail domains.

[0093] In general, in the case of a DNA nucleic acid construct encoding a guide RNA, the U
residues in any of the RNA sequences described herein may be replaced with T
residues, and in the case of a guide RNA construct encoded by a DNA, the T residues may be replaced with U residues.

[0094] Provided herein are compositions comprising one or more guide RNAs or one or more nucleic acids encoding one or more guide RNAs comprising a guide sequence disclosed herein in Table 1A, Table 1B, and Table 5 and throughout the specification.

[0095] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA comprises 17, 18, 19, or 20 contiguous nucleotides of any one of the guide sequences disclosed herein in Table 1A, Table 1B, and Table 5 and throughout the specification.

[0096] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1A, Table 1B, and Table 5 and throughout the specification.

[0097] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guide sequence shown in Table 1A, Table 1B, and Table 5 and throughout the specification.

[0098] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 10, 12, 15, 16, 20, 27, 28, 32, 33, 35, 1001, 1003, 1005, 1010, 1012, 1013, 1016, 1017, and 1018 (for SaCas9). In some embodiments, the spacer sequence is SEQ
ID NO: 10. In some embodiments, the spacer sequence is SEQ ID NO: 12. In some embodiments, the spacer sequence is SEQ ID NO: 15. In some embodiments, the spacer sequence is SEQ ID NO: 16. In some embodiments, the spacer sequence is SEQ ID NO: 20. In some embodiments, the spacer sequence is SEQ ID NO: 27. In some embodiments, the spacer sequence is SEQ ID NO: 28. In some embodiments, the spacer sequence is SEQ ID NO: 32. In some embodiments, the spacer sequence is SEQ ID NO: 33. In some embodiments, the spacer sequence is SEQ ID NO: 35. In some embodiments, the spacer sequence is SEQ ID NO: 1001. In some embodiments, the spacer sequence is SEQ ID NO: 1003. In some embodiments, the spacer sequence is SEQ ID NO:
1005. In some embodiments, the spacer sequence is SEQ ID NO: 1010. In some embodiments, the spacer sequence is SEQ ID NO: 1012. In some embodiments, the spacer sequence is SEQ ID NO:
1013. In some embodiments, the spacer sequence is SEQ ID NO: 1016. In some embodiments, the spacer sequence is SEQ ID NO: 1017. In some embodiments, the spacer sequence is SEQ ID NO:
1018.

[0099] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 3022, 3023, 3028, 3029, 3030, 3031, 3038, 3039, 3052, 3053, 3054, 3055, 3062, 3063, 3064, 3065, 3068, and 3069 (for SaCas9). In some embodiments, the spacer sequence is SEQ
ID NO: 3022. In some embodiments, the spacer sequence is SEQ ID NO: 3023. In some embodiments, the spacer sequence is SEQ ID NO: 3028. In some embodiments, the spacer sequence is SEQ ID NO: 3029. In some embodiments, the spacer sequence is SEQ ID NO:
3030. In some embodiments, the spacer sequence is SEQ ID NO: 3031. In some embodiments, the spacer sequence is SEQ ID NO: 3038. In some embodiments, the spacer sequence is SEQ ID NO:
3039. In some embodiments, the spacer sequence is SEQ ID NO: 3052. In some embodiments, the spacer sequence is SEQ ID NO: 3053. In some embodiments, the spacer sequence is SEQ ID NO:
3054. In some embodiments, the spacer sequence is SEQ ID NO: 3055. In some embodiments, the spacer sequence is SEQ ID NO: 3062. In some embodiments, the spacer sequence is SEQ ID NO:
3063. In some embodiments, the spacer sequence is SEQ ID NO: 3064. In some embodiments, the spacer sequence is SEQ ID NO: 3065. In some embodiments, the spacer sequence is SEQ ID NO:
3068. In some embodiments, the spacer sequence is SEQ ID NO: 3069.

[00100] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 131, 134, 135, 136 ,139, 140, 141, 144, 145, 146, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225 (for SluCas9). In some embodiments, the spacer sequence is SEQ ID NO:
131. In some embodiments, the spacer sequence is SEQ ID NO: 134. In some embodiments, the spacer sequence is SEQ ID NO: 135. In some embodiments, the spacer sequence is SEQ ID NO: 136.
In some embodiments, the spacer sequence is SEQ ID NO: 139. In some embodiments, the spacer sequence is SEQ ID NO: 140. In some embodiments, the spacer sequence is SEQ ID
NO: 141. In some embodiments, the spacer sequence is SEQ ID NO: 144. In some embodiments, the spacer sequence is SEQ ID NO: 145. In some embodiments, the spacer sequence is SEQ ID
NO: 146. In some embodiments, the spacer sequence is SEQ ID NO: 148. In some embodiments, the spacer sequence is SEQ ID NO: 149. In some embodiments, the spacer sequence is SEQ ID
NO: 150. In some embodiments, the spacer sequence is SEQ ID NO: 151. In some embodiments, the spacer sequence is SEQ ID NO: 179. In some embodiments, the spacer sequence is SEQ ID
NO: 184. In some embodiments, the spacer sequence is SEQ ID NO: 201. In some embodiments, the spacer sequence is SEQ ID NO: 223. In some embodiments, the spacer sequence is SEQ ID
NO: 224. In some embodiments, the spacer sequence is SEQ ID NO: 225.

[00101] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 4062, 4063, 4068, 4069, 4070, 4071, 4072, 4073, 4078, 4079, 4088, 4089, 4096, 4097, 4098, 4099, 4100, 4101, 4102, 4103, 4158, 4159, 4168, 4169, 4202, 4203, 4220, 4221, 4246, 4247, 4248, 4249, 4250, 4251 (for SluCas9). In some embodiments, the spacer sequence is SEQ ID
NO: 4062. In some embodiments, the spacer sequence is SEQ ID NO: 4063. In some embodiments, the spacer sequence is SEQ ID NO: 4068. In some embodiments, the spacer sequence is SEQ ID NO:
4069. In some embodiments, the spacer sequence is SEQ ID NO: 4070. In some embodiments, the spacer sequence is SEQ ID NO: 4071. In some embodiments, the spacer sequence is SEQ ID NO:
4072. In some embodiments, the spacer sequence is SEQ ID NO: 4073. In some embodiments, the spacer sequence is SEQ ID NO: 4078. In some embodiments, the spacer sequence is SEQ ID NO:
4079. In some embodiments, the spacer sequence is SEQ ID NO: 4088. In some embodiments, the spacer sequence is SEQ ID NO: 4089. In some embodiments, the spacer sequence is SEQ ID NO:
4096. In some embodiments, the spacer sequence is SEQ ID NO: 4097. In some embodiments, the spacer sequence is SEQ ID NO: 4098. In some embodiments, the spacer sequence is SEQ ID NO:
4099. In some embodiments, the spacer sequence is SEQ ID NO: 4100. In some embodiments, the spacer sequence is SEQ ID NO: 4101. In some embodiments, the spacer sequence is SEQ ID NO:
4102. In some embodiments, the spacer sequence is SEQ ID NO: 4103. In some embodiments, the spacer sequence is SEQ ID NO: 4158. In some embodiments, the spacer sequence is SEQ ID NO:
4159. In some embodiments, the spacer sequence is SEQ ID NO: 4168. In some embodiments, the spacer sequence is SEQ ID NO: 4169. In some embodiments, the spacer sequence is SEQ ID NO:
4202. In some embodiments, the spacer sequence is SEQ ID NO: 4203. In some embodiments, the spacer sequence is SEQ ID NO: 4220. In some embodiments, the spacer sequence is SEQ ID NO:
4221. In some embodiments, the spacer sequence is SEQ ID NO: 4246. In some embodiments, the spacer sequence is SEQ ID NO: 4247. In some embodiments, the spacer sequence is SEQ ID NO:
4248. In some embodiments, the spacer sequence is SEQ ID NO: 4249. In some embodiments, the spacer sequence is SEQ ID NO: 4250. In some embodiments, the spacer sequence is SEQ ID NO:
4251.

[00102] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA further comprises a trRNA. In each composition and method embodiment described herein, the crRNA (comprising the spacer sequence) and trRNA
may be associated as a single RNA (sgRNA) or may be on separate RNAs (dgRNA).
In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.

[00103] In one aspect, a composition is provided comprising a single nucleic acid molecule encoding, or two nucleic acid molecules where one molecule encodes, 1) one or more guide RNA
that comprises a guide sequence selected from any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069; and 2) a SaCas9. In one aspect, a composition is provided comprising a single nucleic acid molecule encoding, or two nucleic acid molecules where one molecule encodes, 1) one or more guide RNA that comprises a guide sequence selected from any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251; and 2) a SluCas9.

[00104] In one aspect, a composition is provided comprising a single nucleic acid molecule encoding, or two nucleic acid molecules where one molecule encodes, 1) one or more guide RNA that comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%
identical to any one of SEQ ID NOs: 1-35, 1000-1078, 3000-3069; and 2) a SaCas9. In one aspect, a composition is provided comprising a single nucleic acid molecule encoding 1) one or more guide RNA that comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251; and 2) a SluCas9.

[00105] In another aspect, a composition is provided comprising a single nucleic acid molecule encoding, or two nucleic acid molecules where one molecule encodes, 1) one or more guide RNA
that comprises a guide sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069; and 2) a SaCas9. In another aspect, a composition is provided comprising a single nucleic acid molecule encoding, or two nucleic acid molecules where one molecule encodes, 1) one or more guide RNA
that comprises a guide sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251; and 2) a SluCas9.

[00106] In any embodiment comprising a nucleic acid molecule encoding a guide RNA and/or a Cas9, the nucleic acid molecule may be a vector. In some embodiments, a composition is provided comprising a single nucleic acid molecule encoding a guide RNA and Cas9, wherein the nucleic acid molecule is a vector.

[00107] Any type of vector, such as any of those described herein, may be used. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a non-integrating viral vector (i.e., that does not insert sequence from the vector into a host chromosome). In some embodiments, the viral vector is an adeno-associated virus vector (AAV), a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the vector comprises a muscle-specific promoter. Exemplary muscle-specific promoters include a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter. See US 2004/0175727 Al;
Wang et al., Expert Opin Drug Del/v. (2014) 11, 345-364; Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. In any of the foregoing embodiments, the vector may be an adeno-associated virus vector (AAV).

[00108] In some embodiments, the muscle specific promoter is the CK8 promoter. The CK8 promoter has the following sequence (SEQ ID NO. 700):

[00109] In some embodiments, the muscle-cell cell specific promoter is a variant of the CK8 promoter, called CK8e. In some embodiments, the size of the CK8e promoter is 436 bp. The CK8e promoter has the following sequence (SEQ ID NO. 701):

[00110] In some embodiments, the vector comprises one or more of a U6, H1, or 7SK promoter.
In some embodiments, the U6 promoter is the human U6 promoter (e.g., the U6L
promoter or U6S
promoter). In some embodiments, the promoter is the murine U6 promoter. In some embodiments, the 7SK promoter is a human 7SK promoter. In some embodiments, the 7SK
promoter is the 7SK1 promoter. In some embodiments, the 7SK promoter is the 7SK2 promoter. In some embodiments, the H1 promoter is a human H1 promoter (e.g., the H1L promoter or the H1S
promoter). In some embodiments, the vector comprises multiple guide sequences, wherein each guide sequence is under the control of a separate promoter. In some embodiments, each of the multiple guide sequences comprises a different sequence. In some embodiments, each of the multiple guide sequences comprise the same sequence (e.g., each of the multiple guide sequences comprise the same spacer sequence). In some embodiments, each of the multiple guide sequences comprises the same spacer sequence and the same scaffold sequence. In some embodiments, each of the multiple guide sequences comprises different spacer sequences and different scaffold sequences. In some embodiments, each of the multiple guide sequences comprises the same spacer sequence, but comprises a different scaffold sequence. In some embodiments, each of the multiple guide sequences comprises different spacer sequences and different scaffold sequences. In some embodiments, each of the separate promoters comprises the same nucleotide sequence (e.g., the U6 promoter sequence). In some embodiments, each of the separate promoters comprises a different nucleotide sequence (e.g., the U6, H1, and/or 7SK promoter sequence).

[00111] In some embodiments, the U6 promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ
ID NO: 702:
cgagtccaac acccgtggga atcccatggg caccatggcc cctcgctcca aaaatgcttt 60 cgcgtcgcgc agacactgct cggtagtttc ggggatcagc gtttgagtaa gagcccgcgt 120 ctgaaccctc cgcgccgccc cggccccagt ggaaagacgc gcaggcaaaa cgcaccacgt 180 gacggagcgt gaccgcgcgc cgagcgcgcg ccaaggtcgg gcaggaagag ggcctatttc 240 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta 300 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc 360 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa 420 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aa 472

[00112] In some embodiments, the H1 promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ
ID NO: 703:
gctcggcgcg cccatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa 60 atgtctttgg atttgggaat cttataagtt ctgtatgaga ccacggta 108

[00113] In some embodiments, the 7SK promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ
ID NO: 704:
tgacggcgcg ccctgcagta tttagcatgc cccacccatc tgcaaggcat tctggatagt 60 gtcaaaacag ccggaaatca agtccgttta tctcaaactt tagcattttg ggaataaatg 120 atatttgcta tgctggttaa attagatttt agttaaattt cctgctgaag ctctagtacg 180 ataagtaact tgacctaagt gtaaagttga gatttccttc aggtttatat agcttgtgcg 240 ccgcctgggt a

[00114] In some embodiments, the U6 promoter is a hU6c promoter and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 705:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG
ATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAG
AAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCAT
ATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC
GAAACACC.

[00115] In some embodiments, the U6 promoter is a variant of the hU6c promoter. In some embodiments, the variant of the hU6c promoter comprises alternative nucleotides as compared to the sequence of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter comprises fewer nucleotides as compared to the 249 nucleotides of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter has fewer nucleotides in the nucleosome binding sequence of the hU6c promoter of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides) the nucleotides corresponding to nucleotides 96-125 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides) the nucleotides corresponding to nucleotides 81-140 of SEQ ID NO:
705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, or 85 nucleotides) the nucleotides corresponding to nucleotides 66-150 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides) the nucleotides corresponding to nucleotides 51-170 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks the nucleotides corresponding to nucleotides 96-125 of SEQ ID
NO: 705. In some embodiments, the variant of the hU6c promoter comprises 129-219 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 219 nucleotides.
In some embodiments, the variant of the hU6c promoter comprises 189 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 159 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 129 nucleotides.

[00116] In some embodiments, the U6 promoter is hU6d30 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 9001:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG
ATAATTGGAATTAATTTGACTGTAAACACAAAGATATAATTTCTTGGGTAGTTTGCAGTT
TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT
TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC.

[00117] In some embodiments, the U6 promoter is hU6d60 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 9002:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG
ATAATTGGAATTAATTTGACGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATA
TGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACG
AAACACC.

[00118] In some embodiments, the U6 promoter is hU6d90 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 9003:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG
ATAATATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT
TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC.

[00119] In some embodiments, the U6 promoter is hU6d120 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 9004:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCGGACTATCAT
ATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC
GAAACACC.

[00120] In some embodiments, the 7SK promoter is a 75K2 promoter and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 706:
CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC
GGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATG
CTGGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTG
ACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTAC
CTC.

[00121] In some embodiments, the 7SK promoter is a variant of the 75K2 promoter. In some embodiments, the variant of the 75K2 promoter comprises alternative nucleotides as compared to the sequence of SEQ ID NO: 706. In some embodiments, the variant of the 75K2 promoter e.g., comprises fewer nucleotides as compared to the 243 nucleotides of SEQ ID NO:
706. In some embodiments, the variant of the 75K2 promoter has fewer nucleotides in the nucleosome binding sequence of the 75K2 promoter of SEQ ID NO: 706. In some embodiments, the variant of the 75K2 promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides) the nucleotides corresponding to nucleotides 95-124 of SEQ ID NO: 706. In some embodiments, the variant of the 75K2 promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides) the nucleotides corresponding to nucleotides 81-140 of SEQ ID NO:
706. In some embodiments, the variant of the 75K2 promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85 or 90 nucleotides) the nucleotides corresponding to nucleotides 67-156 of SEQ ID NO: 706. In some embodiments, the variant of the 75K2 promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides) the nucleotides corresponding to nucleotides 52-171 of SEQ ID NO:
706. In some embodiments, the variant of the 75K2 promoter comprises 123-213 nucleotides.
In some embodiments, the variant of the 75K2 promoter comprises 213 nucleotides. In some embodiments, the variant of the 75K2 promoter comprises 183 nucleotides. In some embodiments, the variant of the 75K2 promoter comprises 153 nucleotides. In some embodiments, the variant of the 75K2 promoter comprises 123 nucleotides.

[00122] In some embodiments, the 7SK promoter is 7SKd30 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 9006:
CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC
GGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTAAATTAGATTTTAGTTAAATTTCCT
GCTGAAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAGG
TTTATATAGCTTGTGCGCCGCTTGGGTACCTC.

[00123] In some embodiments, the 7SK promoter is 7SKd60 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 9007:
CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC
GGAAATCAAGTCCGTTTATCTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTG
ACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTAC
CTC.

[00124] In some embodiments, the 7SK promoter is 7SKd90 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 9008:
CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC
GGAAATAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAG
GTTTATATAGCTTGTGCGCCGCTTGGGTACCTC.

[00125] In some embodiments, the 7SK promoter is 7SKd120 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9009:

[00126] CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAG
CAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTT
GGGTACCTC.

[00127] In some embodiments, the H1 promoter is a Him or mH1 promoter and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to the sequence of SEQ ID NO: 707:
AATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGAT
TTGGGAATCTTATAAGTTCTGTATGAGACCACTCTTTCCC.

[00128] In some embodiments, the Ck8e promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ
ID NO: 701 TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCA
GACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCATGCC
ATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAGGAA
CCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATACAAGGCCATGGGGCTGGGCAAGCTG
CACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTC
TCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTA
TATAACCCAGGGGCACAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAG
ACACTCAGGAGCCAGCCAGC.

[00129] In some embodiments, the vector comprises multiple inverted terminal repeats (ITRs).
These ITRs may be of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or serotype. In some embodiments, the ITRs are of an AAV2 serotype. In some embodiments, the 5' ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 709:
GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG
ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG
CCAACTCCATCACTAGGGGTTCCT.

[00130] In some embodiments, the 3'ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:
710:
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG
CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG
CGAGCGCGCAGAGAGGGA.

[00131] In some embodiments, a vector comprising a single nucleic acid molecule encoding 1) one or more guide RNA comprising any one or more of the spacer sequences of SEQ ID Nos: 1-35, 100-225, 1000-1078, 2000-2116, 3000-3069, or 4000-4251; and 2) a SaCas9 (for SEQ ID Nos: 1-35, 1000-1078, and 3000-3069) or SluCas9 (for SEQ ID NO: 100-225, 2000-2116, and 4000-4251) is provided. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is administered to a subject to treat DMD. In some embodiments, only one vector is needed due to the use of a particular guide sequence that is useful in the context of SaCas9 or SluCas9.

[00132] In some embodiments, the vector comprises a nucleic acid encoding a Cas9 protein (e.g., an SaCas9 or SluCas9 protein) and further comprises a nucleic acid encoding one or more single guide RNA(s). In some embodiments, the nucleic acid encoding the Cas9 protein is under the control of a CK8e promoter. In some embodiments, the nucleic acid encoding the guide RNA sequence is under the control of a hU6c promoter. In some embodiments, the vector is AAV9.

[00133] In some embodiments, the vector comprises multiple nucleic acids encoding more than one guide RNA. In some embodiments, the vector comprises two nucleic acids encoding two guide RNA sequences.

[00134] In some embodiments, the vector comprises a nucleic acid encoding a Cas9 protein (e.g., an SaCas9 protein or SluCas9 protein), a nucleic acid encoding a first guide RNA, and a nucleic acid encoding a second guide RNA. In some embodiments, the vector does not comprise a nucleic acid encoding more than two guide RNAs. In some embodiments, the nucleic acid encoding the first guide RNA is the same as the nucleic acid encoding the second guide RNA. In some embodiments, the nucleic acid encoding the first guide RNA is different from the nucleic acid encoding the second guide RNA. In some embodiments, the vector comprises a single nucleic acid molecule, wherein the single nucleic acid molecule comprises a nucleic acid encoding a Cas9 protein, a nucleic acid encoding a first guide RNA, and a nucleic acid that is the reverse complement to the coding sequence for the second guide RNA. In some embodiments, the vector comprises a single nucleic acid molecule, wherein the single nucleic acid molecule comprises a nucleic acid encoding a Cas9 protein, a nucleic acid that is the reverse complement to the coding sequence for the first guide RNA, and a nucleic acid that is the reverse complement to the coding sequence for the second guide RNA. In some embodiments, the nucleic acid encoding a Cas9 protein (e.g., an SaCas9 or SluCas9 protein) is under the control of the CK8e promoter. In some embodiments, the first guide is under the control of the 75K2 promoter, and the second guide is under the control of the Him promoter. In some embodiments, the first guide is under the control of the Him promoter, and the second guide is under the control of the 75K2 promoter. In some embodiments, the first guide is under the control of the hU6c promoter, and the second guide is under the control of the Him promoter.
In some embodiments, the first guide is under the control of the Him promoter, and the second guide is under the control of the hU6c promoter. In some embodiments, the nucleic acid encoding the Cas9 protein is: a) between the nucleic acids encoding the guide RNAs, b) between the nucleic acids that are the reverse complement to the coding sequences for the guide RNAs, c) between the nucleic acid encoding the first guide RNA and the nucleic acid that is the reverse complement to the coding sequence for the second guide RNA, d) between the nucleic acid encoding the second guide RNA
and the nucleic acid that is the reverse complement to the coding sequence for the first guide RNA, e) 5' to the nucleic acids encoding the guide RNAs, f) 5' to the nucleic acids that are the reverse complements to the coding sequences for the guide RNAs, g) 5' to a nucleic acid encoding one of the guide RNAs and 5' to a nucleic acid that is the reverse complement to the coding sequence for the other guide RNA, h) 3' to the nucleic acids encoding the guide RNAs, i) 3' to the nucleic acids that are the reverse complements to the coding sequences for the guide RNAs, or j) 3' to a nucleic acid encoding one of the guide RNAs and 3' to a nucleic acid that is the reverse complement to the coding sequence for the other guide RNA. In some embodiments, any of the vectors disclosed herein is AAV9. In preferred embodiments, the AAV9 vector is less than 5 kb from ITR to ITR in size, inclusive of both ITRs. In particular embodiments, the AAV9 vector is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.85 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.8 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs.
In further embodiments, the AAV9 vector is less than 4.7 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the vector is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR
in size, inclusive of both ITRs. In some embodiments, the vector is between 4.4-4.85 kb from ITR to ITR
in size, inclusive of both ITRs. In some embodiments, the vector is an AAV9 vector.

[00135] In some embodiments, any of the vectors disclosed herein comprises a nucleic acid encoding at least a first guide RNA and a second guide RNA. In some embodiments, the nucleic acid comprises a spacer-encoding sequence for the first guide RNA, a scaffold-encoding sequence for the first guide RNA, a spacer-encoding sequence for the second guide RNA, and a scaffold-encoding sequence of the second guide RNA. In some embodiments, the spacer-encoding sequence (e.g., encoding any of the spacer sequences disclosed herein) for the first guide RNA
is identical to the spacer-encoding sequence for the second guide RNA. In some embodiments, the spacer-encoding sequence (e.g., encoding any of the spacer sequences disclosed herein) for the first guide RNA is different from the spacer-encoding sequence for the second guide RNA. In some embodiments, the scaffold-encoding sequence for the first guide RNA is identical to the scaffold-encoding sequence for the second guide RNA. In some embodiments, the scaffold-encoding sequence for the first guide RNA is different from the scaffold-encoding sequence for the nucleic acid encoding the second guide RNA. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises a sequence selected from the group consisting of SEQ ID Nos: 901-916, and the scaffold-encoding sequence for the second guide RNA comprises a different sequence selected from the group consisting of SEQ ID Nos: 901-916. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 902. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO:
903. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA
comprises the sequence of SEQ ID NO: 904. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 905. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ
ID NO: 901, and the scaffold-encoding sequence for the second guide RNA
comprises the sequence of SEQ ID NO: 906. In some embodiments, the scaffold-encoding sequence for the first guide RNA
comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 907. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID
NO: 908. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ
ID NO: 901, and the scaffold-encoding sequence for the second guide RNA
comprises the sequence of SEQ ID NO: 909. In some embodiments, the scaffold-encoding sequence for the first guide RNA
comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 910. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID
NO: 911. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ
ID NO: 901, and the scaffold-encoding sequence for the second guide RNA
comprises the sequence of SEQ ID NO: 912. In some embodiments, the scaffold-encoding sequence for the first guide RNA
comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 913. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID
NO: 914. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ
ID NO: 901, and the scaffold-encoding sequence for the second guide RNA
comprises the sequence of SEQ ID NO: 915. In some embodiments, the scaffold-encoding sequence for the first guide RNA
comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 916. In some embodiments, the spacer encoding sequence for the first guide RNA is the same as the spacer-encoding sequence in the second guide RNA, and the scaffold-encoding sequence for the first guide RNA is different from the scaffold-encoding sequence in the nucleic acid encoding the second guide RNA.
The disclosure provides for novel AAV vector configurations. Some examples of these novel AAV
vector configurations are provided herein, and the order of elements in these exemplary vectors are referenced in a 5' to 3' manner with respect to the plus strand. For these configurations, it should be understood that the recited elements may not be directly contiguous, and that one or more nucleotides or one or more additional elements may be present between the recited elements. However, in some embodiments, it is possible that no nucleotides or no additional elements are present between the recited elements. Also, unless otherwise stated, "a promoter for expression of element X" means that the promoter is oriented in a manner to facilitate expression of the recited element X. In addition, unless otherwise stated, references to an "sgRNA scaffold sequence" or "a guide RNA scaffold sequence" are synonymous with "a nucleotide sequence/nucleic acid encoding an sgRNA scaffold sequence" or "a nucleotide sequence/nucleic acid encoding a guide RNA scaffold sequence." In some embodiments, the disclosure provides for a nucleic acid encoding an SaCas9 (e.g., an SaCas9-KKH) or SluCas9. In some embodiments, the nucleic acid encodes for one or more nuclear localization signals (e.g., the 5V40 NLS and/or the c-Myc NLS) on the C-terminus of the encoded SaCas9 or SluCas9. In some embodiments, the nucleic acid encodes for one or more NLSs (e.g., the 5V40 NLS
and/or the c-Myc NLS) on the C-terminus of the encoded SaCas9 or SluCas9, and the nucleic acid does not encode for an NLS on the N-terminus of the encoded SaCas9 or SluCas9.
In some embodiments, the nucleic acid encodes for one or more nuclear localization signals (e.g., the 5V40 NLS and/or the c-Myc NLS) on the N-terminus of the encoded SaCas9 or SluCas9.
In some embodiments, the nucleic acid encodes for one or more NLSs (e.g., the 5V40 NLS
and/or the c-Myc NLS) on the N-terminus of the encoded SaCas9 or SluCas9, and the nucleic acid does not encode for an NLS on the C-terminus of the encoded SaCas9 or SluCas9. In some embodiments, the nucleic acid encodes for one or more nuclear localization signals (e.g., the 5V40 NLS
and/or the c-Myc NLS) on the C-terminus of the encoded SaCas9 or SluCas9 and also encodes for one or more NLSs on the N-terminus of the encoded SaCas9 or SluCas9 (e.g., the 5V40 NLS and/or the c-Myc NLS). In some embodiments, the nucleic acid encodes one NLS. In some embodiments, the nucleic acid encodes two NLSs. In some embodiments, the nucleic acid encodes three NLSs. The one, two, or three NLS may all be C-terminal, N-terminal, or any combination of C- and N-terminal. The NLS may be fused/attached directly to the C- or N-terminus or to another NLS, or may be fused/attached indirectly attached through a linker. In some embodiments, an additional domain may be:
a) fused to the N- or C-terminus of the Cas protein (e.g., a Cas9 protein), b) fused to the N-terminus of an NLS fused to the N-terminus of a Cas protein, or c) fused to the C-terminus of an NLS fused to the C-terminus of a Cas protein, with or without a linker. In some embodiments, an NLS is fused to the N- and/or C-terminus of the Cas protein by means of a linker. In some embodiments, an NLS is fused to the N-terminus of an N-terminally-fused NLS on a Cas protein by means of a linker, and/or an NLS
is fused to the C-terminus of a C-terminally fused NLS on a Cas protein by means of a linker. In some embodiments, the linker is GSVD (SEQ ID NO: 550) or GSGS (SEQ ID NO: 551). In some embodiments, the Cas protein comprises a c-Myc NLS fused to the N-terminus of the Cas protein (or to an N-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises an 5V40 NLS fused to the C-terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises a nucleoplasmin NLS fused to the C-terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N-terminus of the Cas protein, optionally by means of a linker, b) an 5V40 NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) a nucleoplasmin NLS fused to the C-terminus of the 5V40 NLS, optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N-terminus of the Cas protein, optionally by means of a linker, b) a nucleoplasmin NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) an 5V40 NLS fused to the C-terminus of the nucleoplasmin NLS, optionally by means of a linker.

[00136] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, a promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA
scaffold sequence. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA is any of the hU6c promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO:
705. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9001. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9002. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9003. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9004. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA is any of the 75K2 promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9006. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA
comprises SEQ ID NO: 9007. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9008. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO:
9009. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the hU6c promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO:
9001. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9002. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9003. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9004. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the 75K2 promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO:
9006. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9007. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9008. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9009. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the Him promoters disclosed herein. In some embodiments, the promoter for SaCas9 is the CK8e promoter. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS is fused to the SaCas9 with a linker.

[00137] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, an hU6c promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA
scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 15. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 12. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS).
In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00138] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, an 7SK
promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA
scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 15. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 12. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS).
In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00139] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA
scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 15. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 12. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS).
In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00140] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an 7SK promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA
scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 15. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 10 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 12. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS).
In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00141] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9-KKH (e.g., a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ
ID NO: 715 or functional fragments thereof), a polyadenylation sequence, an hU6c promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00142] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9-KKH (e.g., a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ
ID NO: 715 or functional fragments thereof), a polyadenylation sequence, an 7SK promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an SV40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00143] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9-KKH (e.g., a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ
ID NO: 715 or functional fragments thereof), a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00144] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an 7SK promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9-KKH (e.g., a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ
ID NO: 715 or functional fragments thereof), a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 15. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1001 and 16. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 1005. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1003.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1010. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1012. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 12 and 1013. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 1016. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1017 and 16.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1018 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1001. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 15 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1001. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1005 and 1003. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1003 and 16. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1010 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1012 and 12. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 1013 and 12. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 1016 and 10.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1017. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 16 and 1018. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SaCas9 with a linker.

[00145] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SluCas9 (e.g., CK8e), a nucleic acid encoding SluCas9, a polyadenylation sequence, a promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA
scaffold sequence. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA is any of the hU6c promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO:
705. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9001. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9002. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9003. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9004. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA is any of the 75K2 promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 7005. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9006. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA
comprises SEQ ID NO: 9007. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9008. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO:
9009. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the hU6c promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO:
9001. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9002. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9003. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9004. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the 75K2 promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO:
9006. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9007. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9008. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9009. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the Him promoters disclosed herein. In some embodiments, the promoter for SluCas9 is the CK8e promoter. In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS is fused to the SluCas9 with a linker.

[00146] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SluCas9 (e.g., CK8e), a nucleic acid encoding SluCas9, a polyadenylation sequence, an hU6c promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 148 and 134. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 135. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 135. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 136. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 136. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 139 and 131. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 140 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 140 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 141 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 149. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 150. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 131. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 151. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 146 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 134 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
135 and 149. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 135 and 150. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 136 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 136 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 139.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 151 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 148 and 141. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 144. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 144. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 145. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 145. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 148 and 146. In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SluCas9 with a linker.

[00147] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SluCas9 (e.g., CK8e), a nucleic acid encoding SluCas9, a polyadenylation sequence, an 7SK
promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA
scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 148 and 134. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 135. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 135. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 136. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 136. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 139 and 131. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 140 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 140 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 141 and

148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 149. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 150. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 131. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 151. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 146 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 134 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
135 and 149. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 135 and 150. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 136 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 136 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 139.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 151 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 148 and 141. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 144. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 144. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 145. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 145. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 148 and 146. In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SluCas9 with a linker.
[00148] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SluCas9 (e.g., CK8e), a nucleic acid encoding SluCas9, a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 148 and 134. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 135. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 135. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 136. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 136. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 139 and 131. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 140 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 140 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 141 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 149. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 150. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 131. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 151. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 146 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 134 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
135 and 149. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 135 and 150. In some embodiments, the first sgRNA and the second sgRNA

comprise the sequences of SEQ ID NOs: 136 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 136 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 139.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 151 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 148 and 141. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 144. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 144. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 145. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 145. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 148 and 146. In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SluCas9 with a linker.

[00149] In some embodiments, the AAV vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an 7SK promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SluCas9 (e.g., CK8e), a nucleic acid encoding SluCas9, a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 148 and 134. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 135. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 135. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 136. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 136. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 139 and 131. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 140 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 140 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 141 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 149. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 144 and 150. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 131. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 145 and 151. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 146 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 134 and 148. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
135 and 149. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 135 and 150. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 136 and 131. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 136 and 151. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 139.
In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 151 and 140. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 148 and 141. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 149 and 144. In some embodiments, the first sgRNA
and the second sgRNA comprise the sequences of SEQ ID NOs: 150 and 144. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 131 and 145. In some embodiments, the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs:
151 and 145. In some embodiments, the first sgRNA and the second sgRNA
comprise the sequences of SEQ ID NOs: 148 and 146. In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SluCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an 5V40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS
is fused to the SluCas9 with a linker.

[00150] In some embodiments, the disclosure provides for a composition comprising at least two nucleic acids. In some embodiments, the composition comprises at least two nucleic acid molecules, wherein the first nucleic acid molecule comprises a sequence encoding any of the endonucleases disclosed herein (e.g., a SaCas9 or SluCas9), wherein the second nucleic acid molecule encodes a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA are not the same sequence, and wherein the second nucleic acid molecule does not encode an endonuclease.
In some embodiments, the first nucleic acid molecule also encodes a copy of the first guide RNA and a copy of the second guide RNA. In some embodiments, the first nucleic acid molecule does not encode any guide RNAs. In some embodiments, the second nucleic acid molecule encodes two copies of the first guide RNA and two copies of the second guide RNA. In some embodiments, the second nucleic molecule encodes two copies of the first guide RNA, and one copy of the second guide RNA. In some embodiments, the second nucleic acid molecule encodes one copy of the first guide RNA, and two copies of the second guide RNA. In some embodiments, the second nucleic acid molecule comprises two copies of the first guide RNA, and three copies of the second guide RNA. In some embodiments, the second nucleic acid molecule comprises three copies of the first guide RNA, and two copies of the second guide RNA. In some embodiments, the second nucleic acid molecule encodes three copies of the first guide RNA and three copies of the second guide RNA. In some embodiments, the first nucleic acid molecule comprises from 5' to 3' with respect to the plus strand:
the reverse complement of a first guide RNA scaffold sequence, the reverse complement of a nucleotide sequence encoding the first guide RNA sequence, the reverse complement of a promoter for expression of the nucleotide sequence encoding the first guide RNA
sequence, a promoter for expression of a nucleotide sequence encoding the endonuclease, a nucleotide sequence encoding an endonuclease, a polyadenylation sequence, a promoter for expression of the second guide RNA in the same direction as the promoter for the endonuclease, the second guide RNA
sequence, and a second guide RNA scaffold sequence. In some embodiments, the promoter for expression of the nucleotide sequence encoding the first guide RNA sequence in the first nucleic acid molecule is a U6 promoter and the promoter for expression of the nucleotide sequence encoding the second guide RNA in the first nucleic acid molecule is a U6 promoter. In some embodiments, the first nucleic acid molecule encodes a Staphylococcus aureus Cas9 (SaCas9) endonuclease, and the first guide RNA comprises the first sequence and the second guide RNA comprises the second sequence from any one the following pairs of sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;
1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12;
1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018. In some embodiments, the first guide RNA
comprises the sequence of SEQ ID NO: 12 and the second guide RNA comprises the sequence of SEQ ID NO: 1013. In some embodiments, the first guide RNA comprises the sequence of SEQ ID
NO: 1013 and the second guide RNA comprises the sequence of SEQ ID NO: 12. In some embodiments, the first nucleic acid molecule encodes a Staphylococcus lugdunensis (SluCas9) endonuclease, and wherein the first guide RNA comprises the first sequence and the second guide RNAs comprises the second sequence from any one the following pairs of sequences: SEQ ID NOs:
148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131;
139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139;

131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145;

151 and 145; and 148 and 146. In some embodiments, a) the first guide RNA comprises the sequence of SEQ ID NO:
148 and the second guide RNA comprises the sequence of SEQ ID NO: 134; b) the second guide RNA comprises the sequence of SEQ ID NO: 145 and the second guide RNA
comprises the sequence of SEQ ID NO: 131, c) the first guide RNA comprises the sequence of SEQ ID NO:
134 and the second guide RNA comprises the sequence of SEQ ID NO: 148; d) the second guide RNA comprises the sequence of SEQ ID NO: 131 and the second guide RNA comprises the sequence of SEQ ID NO:
145.
[00151] In some embodiments, the first nucleic acid molecule is in a first vector (e.g., AAV9), and the second nucleic acid is in a separate second vector. In some embodiments, the first vector is AAV9. In some embodiments, the second vector is AAV9. In preferred embodiments, the AAV9 vector is less than 5 kb from ITR to ITR in size, inclusive of both ITRs. In particular embodiments, the AAV9 vector is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.85 kb from ITR to ITR in size, inclusive of both ITRs.
In further embodiments, the AAV9 vector is less than 4.8 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.7 kb from ITR to ITR
in size, inclusive of both ITRs. In some embodiments, the second vector comprises from 5' to 3' with respect to the plus strand: a promoter for expression of a first copy of a first guide RNA (e.g., a U6 promoter), a first copy of a nucleotide sequence encoding a first guide RNA, a first copy of a nucleotide sequence encoding a first guide RNA scaffold, a promoter for expression of a second copy of the first guide RNA (e.g., a H1 promoter), a second copy of the nucleotide sequence encoding the first guide RNA, a second copy of the nucleotide sequence encoding the first guide RNA scaffold, a promoter for expression of a second guide RNA (e.g., a 7SK promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA scaffold. In some embodiments, the second vector comprises from 5' to 3' with respect to the plus strand: a promoter for expression of a first guide RNA (e.g., a U6 promoter), a nucleotide sequence encoding a first guide RNA, a nucleotide sequence encoding a first guide RNA scaffold, a promoter for expression of a second guide RNA (e.g., a 7SK promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA scaffold. In some embodiments, the second vector comprises a stuffer sequence (e.g., a 3'UTR
desmin sequence) between the nucleotide sequence encoding the first guide scaffold sequence and the promoter for expression of the second guide sequence. In some embodiments, the second vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a scaffold for a first guide RNA, the reverse complement of a nucleotide sequence encoding a first guide RNA, the reverse complement of a promoter for expression of the first guide RNA (e.g., a U6c promoter), a promoter for expression of a second guide RNA (e.g., a U6c promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA
scaffold. In some embodiments, the second vector comprises a stuffer sequence (e.g., a 3'UTR
desmin sequence) between the reverse complement of the promoter for expression of the first guide RNA and the promoter for expression of the second guide RNA. In some embodiments, the second vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first copy of a first guide RNA scaffold, the reverse complement of a nucleotide sequence encoding a first copy of the first guide RNA, the reverse complement of a promoter for expression of the first copy of the first guide RNA (e.g., a 7SK2 promoter), the reverse complement of a second copy of the nucleotide sequence encoding the first guide RNA
scaffold, the reverse complement of a second copy of the nucleotide sequence encoding the first guide RNA, the reverse complement of a promoter for expression of the second copy of the nucleotide sequence encoding the first guide RNA (e.g., a hU6c promoter), a promoter for expression of a first copy of a second guide RNA (e.g., a hU6c promoter), a first copy of a nucleotide sequence encoding a second guide RNA, a first copy of a nucleotide sequence encoding a second guide RNA scaffold, a promoter for expression of a second copy of the second guide RNA (e.g., a 7Sk2 promoter), a second copy of the nucleotide sequence encoding the second guide RNA, and a second copy of the nucleotide sequence encoding the second guide RNA scaffold. In some embodiments, the second vector comprises a stuffer sequence (e.g., a 3 'UTR desmin sequence) between the reverse complement of the promoter for expression of the second copy of the first guide RNA and the promoter for expression of the first copy of the second guide RNA. In some embodiments, the second vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first copy of a first guide RNA scaffold, the reverse complement of a first copy of a nucleotide sequence encoding the first guide RNA, the reverse complement of a promoter for expression of the first copy of the first guide RNA (e.g., a 7SK2 promoter), the reverse complement of a first copy of a nucleotide sequence encoding a second guide RNA scaffold, the reverse complement of a nucleotide sequence encoding the first copy of the second guide RNA, the reverse complement of a promoter for expression of the first copy of the second guide RNA (e.g., a hU6c promoter), a promoter for expression of a second copy of the second guide RNA (e.g., a hU6c promoter), a second copy of the nucleotide sequence encoding the second guide RNA, a second copy of the nucleotide sequence encoding the second guide RNA scaffold, a promoter for expression of a second copy of the first guide RNA (e.g., a 7SK2 promoter), a second copy of the nucleotide sequence encoding the first guide RNA, and a second copy of the nucleotide sequence encoding the first guide RNA scaffold. In some embodiments, the second vector comprises a stuffer sequence (e.g., a 3'UTR desmin sequence) between the reverse complement of the promoter for expression of the first copy of the second guide RNA and the promoter for expression of the second copy of the first guide RNA. In some embodiments, the second vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first guide RNA scaffold, the reverse complement of a nucleotide sequence encoding a first guide RNA, the reverse complement of a promoter for expression of a first guide RNA (e.g., a hU6c promoter), a promoter for expression of a second guide RNA
(e.g., a hU6c promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA scaffold. In some embodiments, the second vector comprises a stuffer sequence (e.g., a 3'UTR desmin sequence) between the reverse complement of the promoter for expression of the first guide RNA and the promoter for expression of the second guide RNA.
In particular embodiments, the first guide RNA is different from the second guide RNA. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 12 and the second guide RNA comprises the sequence of SEQ ID NO: 1013. In some embodiments, the first guide RNA
comprises the sequence of SEQ ID NO: 1013 and the second guide RNA comprises the sequence of SEQ ID NO:
12. In some embodiments, a) the first guide RNA comprises the sequence of SEQ
ID NO: 148 and the second guide RNA comprises the sequence of SEQ ID NO: 134; b) the second guide RNA
comprises the sequence of SEQ ID NO: 145 and the second guide RNA comprises the sequence of SEQ ID NO: 131; c) the first guide RNA comprises the sequence of SEQ ID NO:
134 and the second guide RNA comprises the sequence of SEQ ID NO: 148; or d) the second guide RNA
comprises the sequence of SEQ ID NO: 131 and the second guide RNA comprises the sequence of SEQ ID NO:
145. In some embodiments, the scaffold for the first guide RNA comprises the sequence of SEQ ID
NO: 901. In some embodiments, the scaffold for the second guide RNA comprises the sequence of SEQ ID NO: 901. In some embodiments, any of the second vectors comprises a stuffer sequence. In some embodiments, the stuffer sequence is a 3'UTR sequence. In some embodiments, the 3'UTR
desmin sequence comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 552 (taattaacagatttgt taatgaagaggaaatatacaaatattaatgaacaaaatagtgtgtttagaacaagactcacatacaggagacatacaca tgttaaaggaggcattaga atagtggggaaaacttacaaattcagtatagggtaggagcactagatagctaagaggtggggtagggggcagagtgaag gctgtctctcttttact ccttctaaaaatattccagtggatcaaagatgtaggaaacgatggatccatgtaggcctcagccttcaagtactcctct gagatttcagggattatcctt taaaggagtcaggaagagggtagagatcacaaagataataagctcagacagtagtatgttaaataaacctcaggaggtt ttagtcttaaggtccttaa tctgaccttccaaactgacttaccagaaacttccaaaagcctctc) or fragments thereof

[00152] In some embodiments, if the composition comprises one or more nucleic acids encoding an RNA-targeted endonuclease and one or more guide RNAs, the one or more nucleic acids are designed such that they express the one or more guide RNAs at an equivalent or higher level (e.g., a greater number of expressed transgene copies) as compared to the expression level of the RNA-targeted endonuclease. In some embodiments, the one or more nucleic acids are designed such that they express (e.g., on average in 100 cells) the one or more guide RNAs at least a 1.1, 1.2, 1.3, 1.4, or 1.5 times higher level (e.g., a greater number of expressed transgene copies) as compared to the expression level of the RNA-targeted endonuclease. In some embodiments, the one or more nucleic acids are designed such that they express the one or more guide RNAs at 1.01-1.5, 1.01-1.4, 1.01-1.3, 1.01-1.2, 1.01-1.1, 1.1-2.0, 1.1-1.8, 1.1-1.6, 1.1-1.4, 1.1-1.3, 1.2-2.0, 1.2-1.8, 1.2-1.6, 1.2-1.4, 1.4-2.0, 1.4-1.8, 1.4-1.6, 1.6-2.0, 1.6-1.8, or 1.8-2.0 times higher level (e.g., a greater number of expressed transgene copies) as compared to the expression level of the RNA-targeted endonuclease. In some embodiments, the one or more guide RNAs are designed to express a higher level than the RNA-targeted endonuclease by: a) utilizing one or more regulatory elements (e.g., promoters or enhancers) that express the one or more guide RNAs at a higher level as compared to the regulatory elements (e.g., promoters or enhancers) for expression of the RNA-targeted endonuclease; and/or b) expressing more copies of one or more of the guide RNAs as compared to the number of copies of the RNA-targeted endonuclease (e.g., 2x or 3x as many copies of the nucleotide sequences encoding the one or more guide RNAs as compared to the number of copies of the nucleotide sequences encoding the RNA-targeted endonuclease). For example, in some embodiments, the composition comprises multiple nucleic acid molecules (e.g., in multiple vectors), wherein for every nucleotide sequence encoding an RNA-targeted endonuclease in the nucleic acid molecules in the composition, there are two or three copies of the nucleotide sequence encoding the guide RNA in the nucleic acid molecules in the composition. In some embodiments, the composition comprises a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA are not the same (e.g., any of the guide RNA pairs disclosed herein), and for every nucleotide sequence encoding an RNA-targeted endonuclease in the nucleic acid molecules in the composition, there are two or three copies of the nucleotide sequence encoding the first guide RNA and/or the second guide RNA.

[00153] In some embodiments, any of the nucleic acids disclosed herein encodes an RNA-targeted endonuclease. In some embodiments, the RNA-targeted endonuclease has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-targeted endonuclease comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, e.g., the list in the next paragraph), and modified (e.g., engineered or mutant) versions thereof See, e.g., US2016/0312198 Al; US 2016/0312199 Al. In particular embodiments, the RNA-targeted endonuclease is a type II
CRISPR Cas enzyme. Other examples of Cas nucleases include a Csm or Cmr complex of a type III
CRISPR system or the Cas10, Csml, or Cmr2 subunit thereof; and a Cascade complex of a type I
CRISPR system, or the Cas3 subunit thereof In some embodiments, the Cas nuclease may be from a Type-IA, Type-JIB, or Type-IIC system. For discussion of various CRISPR
systems and Cas nucleases see, e.g., Makarova et al., NAT. REV. MICROBIOL. 9:467-477 (2011);
Makarova et al., NAT.
REV. MICROBIOL, 13: 722-36 (2015); Shmakov et al., MOLECULAR CELL, 60:385-397 (2015).

[00154] Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyo genes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Paste urella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium rose urn, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenifireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denficola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaficum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Fine goldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromafium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, and Acaryochloris marina.

[00155] In some embodiments, the nucleic acid encoding SaCas9 encodes an SaCas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 711:
KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI
QRVKKLLFDYNLLTDHSEL SGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE
DTGNEL STKEQI SRN SKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQK
AYHQLDQ SFIDTYIDLLETRRTYYE GP GEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA
YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY
RVT ST GKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQ S SEDIQEELTNLNSELTQEE
IEQI SNLKGYTGTHNL SLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDL SQQKEIPTTLVDD
FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKN SKDAQKMINEMQKRNRQTNERIEEIIR
TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL
VKQEENSKKGNRTPFQYLS S SD SKI SYETFKKHILNL AKGKGRI SKTKKEYLLEERDINRF SVQ
KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFT SFLRRKWKFKKERNKGYK
HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAE SMPEIETEQEYKEIFITPHQIKHI
KDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP
EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN
KLNAHLDITDDYPNSRNKVVKL SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC
YEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN
DKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00156] In some embodiments, the nucleic acid encoding SaCas9 comprises the nucleic acid of SEQ ID NO: 9014:

[00157] AAGCGCAATTACATCCTGGGCCTGGATATCGGCATCACCTCCGTGGGCTACG
GCATCATCGACTATGAGACACGGGATGTGATCGACGCCGGCGTGAGACTGTTCAAGGAG
GCCAACGTGGAGAACAATGAGGGCCGGCGGAGCAAGAGGGGAGCAAGGCGCCTGAAGC
GGAGAAGGCGCCACAGAATCCAGAGAGTGAAGAAGCTGCTGTTCGATTACAACCTGCTG
ACCGACCACTCCGAGCTGTCTGGCATCAATCCTTATGAGGCCCGGGTGAAGGGCCTGTCC
CAGAAGCTGTCTGAGGAGGAGTTTTCTGCCGCCCTGCTGCACCTGGCAAAGAGGAGAGG
CGTGCACAACGTGAATGAGGTGGAGGAGGACACCGGCAACGAGCTGAGCACAAAGGAG
CAGATCAGCCGCAATTCCAAGGCCCTGGAGGAGAAGTATGTGGCCGAGCTGCAGCTGGA
GCGGCTGAAGAAGGATGGCGAGGTGAGGGGCTCCATCAATCGCTTCAAGACCTCTGACT
ACGTGAAGGAGGCCAAGCAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGATCAG
AGCTTTATCGATACATATATCGACCTGCTGGAGACCAGGCGCACATACTATGAGGGACC
AGGAGAGGGCTCCCCCTTCGGCTGGAAGGACATCAAGGAGTGGTACGAGATGCTGATGG
GCCACTGCACCTATTTTCCAGAGGAGCTGAGATCCGTGAAGTACGCCTATAACGCCGATC
TGTACAACGCCCTGAATGACCTGAACAACCTGGTCATCACCAGGGATGAGAACGAGAAG
CTGGAGTACTATGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGAAGAAGAAGCC
TACACTGAAGCAGATCGCCAAGGAGATCCTGGTGAACGAGGAGGACATCAAGGGCTACC
GCGTGACCAGCACAGGCAAGCCAGAGTTCACCAATCTGAAGGTGTATCACGATATCAAG
GACATCACAGCCCGGAAGGAGATCATCGAGAACGCCGAGCTGCTGGATCAGATCGCCAA
GATCCTGACCATCTATCAGAGCTCCGAGGACATCCAGGAGGAGCTGACCAACCTGAATA
GCGAGCTGACACAGGAGGAGATCGAGCAGATCAGCAATCTGAAGGGCTACACCGGCAC
ACACAACCTGTCCCTGAAGGCCATCAATCTGATCCTGGATGAGCTGTGGCACACAAACG
ACAATCAGATCGCCATCTTTAACAGGCTGAAGCTGGTGCCAAAGAAGGTGGACCTGAGC
CAGCAGAAGGAGATCCCAACCACACTGGTGGACGATTTCATCCTGTCCCCCGTGGTGAA
GCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGC
CCAATGATATCATCATCGAGCTGGCCAGGGAGAAGAACTCTAAGGACGCCCAGAAGATG
ATCAATGAGATGCAGAAGAGGAACCGCCAGACCAATGAGCGGATCGAGGAGATCATCA
GAACCACAGGCAAGGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGATAT
GCAGGAGGGCAAGTGTCTGTATAGCCTGGAGGCCATCCCTCTGGAGGACCTGCTGAACA
ATCCATTCAACTACGAGGTGGATCACATCATCCCCCGGAGCGTGAGCTTCGACAATTCCT
TTAACAATAAGGTGCTGGTGAAGCAGGAGGAGAACTCTAAGAAGGGCAATAGGACCCCT
TTCCAGTACCTGTCTAGCTCCGATTCTAAGATCAGCTACGAGACCTTCAAGAAGCACATC
CTGAATCTGGCCAAGGGCAAGGGCCGCATCTCTAAGACCAAGAAGGAGTACCTGCTGGA
GGAGCGGGACATCAACAGATTCAGCGTGCAGAAGGACTTCATCAACCGGAATCTGGTGG
ACACCAGATACGCCACACGCGGCCTGATGAATCTGCTGCGGTCCTATTTCAGAGTGAACA
ATCTGGATGTGAAGGTGAAGAGCATCAACGGCGGCTTCACCTCCTTTCTGCGGAGAAAG
TGGAAGTTTAAGAAGGAGAGAAACAAGGGCTATAAGCACCACGCCGAGGATGCCCTGAT
CATCGCCAATGCCGACTTCATCTTTAAGGAGTGGAAGAAGCTGGACAAGGCCAAGAAAG

TGATGGAGAACCAGATGTTCGAGGAGAAGCAGGCCGAGAGCATGCCCGAGATCGAGAC
CGAGCAGGAGTACAAGGAGATTTTCATCACACCTCACCAGATCAAGCACATCAAGGACT
TCAAGGACTACAAGTATTCCCACAGGGTGGATAAGAAGCCCAACCGCGAGCTGATCAAT
GACACCCTGTATTCTACAAGGAAGGACGATAAGGGCAATACCCTGATCGTGAACAATCT
GAACGGCCTGTACGACAAGGATAATGACAAGCTGAAGAAGCTGATCAACAAGAGCCCC
GAGAAGCTGCTGATGTACCACCACGATCCTCAGACATATCAGAAGCTGAAGCTGATCAT
GGAGCAGTACGGCGACGAGAAGAACCCACTGTATAAGTACTATGAGGAGACCGGCAACT
ACCTGACAAAGTATTCCAAGAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTAT
GGCAACAAGCTGAATGCCCACCTGGACATCACCGACGATTACCCCAACAGCCGGAATAA
GGTGGTGAAGCTGAGCCTGAAGCCATACAGGTTCGACGTGTACCTGGACAACGGCGTGT
ATAAGTTTGTGACAGTGAAGAATCTGGATGTGATCAAGAAGGAGAACTACTATGAAGTG
AATAGCAAGTGCTACGAGGAGGCCAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGT
TCATCGCCTCTTTTTACAACAATGACCTGATCAAGATCAATGGCGAGCTGTATAGAGTGA
TCGGCGTGAACAATGATCTGCTGAACCGCATCGAAGTGAATATGATCGACATCACCTACC
GGGAGTATCTGGAGAACATGAATGATAAGAGGCCCCCTCGCATCATCAAGACCATCGCC
TCTAAGACACAGAGCATCAAGAAGTACTCTACAGACATCCTGGGCAACCTGTATGAGGT
GAAGAGCAAGAAGCACCCTCAGATCATCAAGAAGGGC.

[00158] In some embodiments comprising a nucleic acid encoding SaCas9, the SaCas9 comprises an amino acid sequence of SEQ ID NO: 711.

[00159] In some embodiments, the SaCas9 is a variant of the amino acid sequence of SEQ ID NO:
711. In some embodiments, the SaCas9 comprises an amino acid other than an E
at the position corresponding to position 781 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 967 of SEQ ID
NO: 711. In some embodiments, the SaCas9 comprises an H at the position corresponding to position 1014 of SEQ ID
NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711; an amino acid other than an N
at the position corresponding to position 967 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 711; a K at the position corresponding to position 967 of SEQ ID NO: 711; and an H at the position corresponding to position 1014 of SEQ
ID NO: 711.

[00160] In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an N at the position corresponding to position 412 of SEQ ID NO:
711. In some embodiments, the SaCas9 comprises an amino acid other than an N
at the position corresponding to position 418 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711; an amino acid other than an N
at the position corresponding to position 412 of SEQ ID NO: 711; an amino acid other than an N
at the position corresponding to position 418 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 244 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 412 of SEQ ID
NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 418 of SEQ ID
NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A
at the position corresponding to position 244 of SEQ ID NO: 711; an A at the position corresponding to position 412 of SEQ ID NO: 711; an A at the position corresponding to position 418 of SEQ
ID NO: 711; and an A
at the position corresponding to position 653 of SEQ ID NO: 711.

[00161] In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 412 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 418 of SEQ ID NO: 711; an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711; an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 244 of SEQ ID NO:
711; an A at the position corresponding to position 412 of SEQ ID NO: 711; an A at the position corresponding to position 418 of SEQ ID NO: 711; an A at the position corresponding to position 653 of SEQ
ID NO: 711; a K at the position corresponding to position 781 of SEQ ID NO: 711; a K at the position corresponding to position 967 of SEQ ID NO: 711; and an H at the position corresponding to position 1014 of SEQ ID
NO: 711.

[00162] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 715 (designated herein as SaCas9-KKH or SACAS9KKH):
KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI
QRVKKLLFDYNLLTDHSEL SGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE
DTGNEL STKEQI SRN SKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQK

AYHQLDQ SFIDTYIDLLETRRTYYEGP GEGSPF GWKDIKEWYEMLM GHCTYFPEELRSVKYA
YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY
RVT ST GKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQ S SEDIQEELTNLNSELTQEE
IEQI SNLKGYTGTHNL SLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDL SQQKEIPTTLVDD
FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKN SKDAQKMINEMQKRNRQTNERIEEIIR
TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL
VKQEENSKKGNRTPFQYLS S SD SKI SYETFKKHILNL AKGKGRI SKTKKEYLLEERDINRF SVQ
KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFT SFLRRKWKFKKERNKGYK
HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAE SMPEIETEQEYKEIFITPHQIKHI
KDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP
EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET GNYLTKY SKKDNGP VIKKIKYYGN
KLNAHLDITDDYPNSRNKVVKL SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC
YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN
DKRPPHIIKTIASKTQ SIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00163] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 716 (designated herein as SaCas9-HF):
KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI
QRVKKLLFDYNLLTDHSEL SGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE
DTGNEL STKEQI SRN SKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQK
AYHQLDQ SFIDTYIDLLETRRTYYE GP GEGSPFGWKDIKEWYEMLMGHCTYFPEELASVKYA
YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY
RVT ST GKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQ S SEDIQEELTNLNSELTQEE
IEQI SNLKGYTGTHNL SLKAINLILDELWHTNDAQIAIFARLKLVPKKVDL SQQKEIPTTLVDD
FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKN SKDAQKMINEMQKRNRQTNERIEEIIR
TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL
VKQEENSKKGNRTPFQYLS S SD SKI SYETFKKHILNL AKGKGRI SKTKKEYLLEERDINRF SVQ
KDFINRNLVDTRYATAGLMNLLRSYFRVNNLD VKVKSINGGFT SFLRRKWKFKKERNKGYK
HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAE SMPEIETEQEYKEIFITPHQIKHI
KDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP
EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET GNYLTKY SKKDNGP VIKKIKYYGN
KLNAHLDITDDYPNSRNKVVKL SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC
YEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN
DKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00164] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 717 (designated herein as SaCas9-KKH-HF):

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI
QRVKKLLFDYNLLTDHSEL SGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE
DTGNEL STKEQI SRN SKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQK
AYHQLDQ SFIDTYIDLLETRRTYYE GP GEGSPFGWKDIKEWYEMLMGHCTYFPEELASVKYA
YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY
RVT ST GKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQ S SEDIQEELTNLNSELTQEE
IEQI SNLKGYTGTHNL SLKAINLILDELWHTNDAQIAIFARLKLVPKKVDL SQQKEIPTTLVDD
FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKN SKDAQKMINEMQKRNRQTNERIEEIIR
TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL
VKQEENSKKGNRTPFQYLS S SD SKI SYETFKKHILNL AKGKGRI SKTKKEYLLEERDINRF SVQ
KDFINRNLVDTRYATAGLMNLLRSYFRVNNLD VKVKSINGGFT SFLRRKWKFKKERNKGYK
HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAE SMPEIETEQEYKEIFITPHQIKHI
KDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP
EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN
KLNAHLDITDDYPNSRNKVVKL SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC
YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN
DKRPPHIIKTIASKTQ SIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00165] In some embodiments, the nucleic acid encoding SluCas9 encodes a SluCas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 712:
NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE
RVKKLLEDYNLLDQSQIPQ STNPYAIRVKGL SEAL SKDELVIALLHIAKRRGIHKIDVID SNDD
VGNEL STKEQLNKN SKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH
QLDENFINKYIELVEMRREYFE GP GKGSPYGWEGD PKAWYETLMGHCTYFPDELRSVKYAY
SADLFNALNDLNNLVIQRD GL S KLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI
TKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKD SIKSKLTELDILLNEEDK
ENIAQLTGYTGTHRL SLKCIRLVLEEQWYS SRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEF
IL SPVVKRTFGQAINLINKIIEKY GVPEDIIIELARENN SKDKQKFINEMQKKNENTRKRINEII G
KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV
LVKQSEN SKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE
VQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH
GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVD SEDNYSEMFIIPKQVQ
DIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE
KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK
LGSHLDVTHQFKS STKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK
LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP
RIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00166] In some embodiments, the S1uCas9 is a variant of the amino acid sequence of SEQ ID
NO: 712. In some embodiments, the S1uCas9 comprises an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises a K at the position corresponding to position 781 of SEQ
ID NO: 712. In some embodiments, the S1uCas9 comprises a K at the position corresponding to position 966 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an H at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises a K at the position corresponding to position 781 of SEQ
ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO:
712; and an H at the position corresponding to position 1013 of SEQ ID NO: 712.

[00167] In some embodiments, the S1uCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO:
712. In some embodiments, the S1uCas9 comprises an amino acid other than a T
at the position corresponding to position 420 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712; an amino acid other than an N
at the position corresponding to position 414 of SEQ ID NO: 712; an amino acid other than a T
at the position corresponding to position 420 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an A at the position corresponding to position 414 of SEQ ID
NO: 712. In some embodiments, the S1uCas9 comprises an A at the position corresponding to position 420 of SEQ ID
NO: 712. In some embodiments, the S1uCas9 comprises an A at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712; an A at the position corresponding to position 414 of SEQ ID NO: 712; an A at the position corresponding to position 420 of SEQ
ID NO: 712; and an A
at the position corresponding to position 655 of SEQ ID NO: 712.

[00168] In some embodiments, the S1uCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712; an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO: 712; an amino acid other than a T at the position corresponding to position 420 of SEQ ID NO: 712; an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712; an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712; an A at the position corresponding to position 414 of SEQ ID NO: 712; an A at the position corresponding to position 420 of SEQ
ID NO: 712; an A at the position corresponding to position 655 of SEQ ID NO: 712; a K at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO:
712; and an H at the position corresponding to position 1013 of SEQ ID NO:
712.

[00169] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 718 (designated herein as SluCas9-KH or SLUCAS9KH):
NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE
RVKKLLEDYNLLDQSQIPQ STNPYAIRVKGL SEAL SKDELVIALLHIAKRRGIHKIDVID SNDD
VGNEL STKEQLNKN SKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH
QLDENFINKYIELVEMRREYFE GP GKGSPYGWEGD PKAWYETLMGHCTYFPDELRSVKYAY
SADLFNALNDLNNLVIQRD GL S KLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI
TKS GKPQFTEFKLYHD LKSVLFDQ SILENEDVLDQIAEILTIYQDKD SIKSKLTELDILLNEEDK
ENIAQLTGYTGTHRL SLKCIRLVLEEQWYS SRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEF
IL SPVVKRTFGQAINLINKIIEKY GVPEDIIIELARENN SKDKQKFINEMQKKNENTRKRINEII G
KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV
LVKQSEN SKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE
VQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH
GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVD SEDNYSEMFIIPKQVQ
DIKDFRNFKYSHRVDKKPNRKLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE
KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK
LGSHLDVTHQFKS STKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK
LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP
HIKKTIGKKVN SIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00170] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 719 (designated herein as SluCas9-HF):
NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE
RVKKLLEDYNLLDQSQIPQ STNPYAIRVKGL SEAL SKDELVIALLHIAKRRGIHKIDVID SNDD
VGNEL STKEQLNKN SKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH
QLDENFINKYIELVEMRREYFEGP GKGSPYGWEGDPKAWYETLMGHCTYFPDELASVKYAY
SADLFNALNDLNNLVIQRD GL S KLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI
TKS GKPQFTEFKLYHD LKSVLFDQ SILENEDVLDQIAEILTIYQDKD SIKSKLTELDILLNEEDK

ENIAQLTGYTGTHRL SLKCIRLVLEEQWYS SRAQMEIFAHLNIKPKKINLTAANKIPKAMIDEF
IL SPVVKRTFGQAINLINKIIEKY GVPEDIIIELARENN SKDKQKFINEMQKKNENTRKRINEII G
KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV
LVKQSEN SKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE
VQKEFINRNLVDTRYATAELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH
GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVD SEDNYSEMFIIPKQVQ
DIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE
KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK
LGSHLDVTHQFKS STKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK
LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP
RIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00171] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 720 (designated herein as SluCas9-HF-KH):
NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE
RVKKLLEDYNLLDQSQIPQ STNPYAIRVKGL SEAL SKDELVIALLHIAKRRGIHKIDVID SNDD
VGNEL STKEQLNKN SKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH
QLDENFINKYIELVEMRREYFEGP GKGSPYGWEGDPKAWYETLMGHCTYFPDELASVKYAY
SADLFNALNDLNNLVIQRD GL S KLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI
TKS GKPQFTEFKLYHD LKSVLFDQ SILENEDVLDQIAEILTIYQDKD SIKSKLTELDILLNEEDK
ENIAQLTGYTGTHRL SLKCIRLVLEEQWYS SRAQMEIFAHLNIKPKKINLTAANKIPKAMIDEF
IL SPVVKRTFGQAINLINKIIEKY GVPEDIIIELARENN SKDKQKFINEMQKKNENTRKRINEII G
KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV
LVKQSEN SKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE
VQKEFINRNLVDTRYATAELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH
GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVD SEDNYSEMFIIPKQVQ
DIKDFRNFKYSHRVDKKPNRKLINDTLY STRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE
KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK
LGSHLDVTHQFKS STKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK
LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP
HIKKTIGKKVN SIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00172] In some embodiments, the Cas protein is any of the engineered Cas proteins disclosed in Schmidt et al., 2021, Nature Communications, "Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases."

[00173] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7021 (designated herein as sRGN1):

MNQKFILGLDIGIT SVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
DRVKHLLAEYDLLDLTNIPKSTNPYQTRVKGLNEKL SKDELVIALLHIAKRRGIHNVDVAAD
KEETA SD SL STKD QINKNAKFLE SRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDT
QMQYYPEIDETFKEKYI SLVETRREYFEGP GKGSPFGWE GNIKKWFEQMMGHCTYFPEELRS
VKYSYSAELFNALNDLNNLVITRDEDAKLNYGEKFQIIENVFKQKKTPNLKQIAIEIGVHETEI
KGYRVNKS GTPEFTEFKLYHDLKSIVFDK SILENEAILDQIAEILTIYQDEQ SIKEELNKLPEILN
EQDKAEIAKLIGYNGTHRLSLKCIHLINEELWQT SRNQMEIFNYLNIKPNKVDLSEQNKIPKD
MVNDFIL SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRI
NEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLKDIPLEDLLRNPNNYDIDHIIPRSVSFDD SM
HNKVLVRREQNAKKNNQTPYQYLT SGYADIKYSVFKQHVLNLAENKDRMTKKKREYLLEE
RDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKF
KKERNHGYKHHAEDALIIANADFLFKENKKLKAVN SVLEKPEIETKQLDIQVD SEDNYSEMFI
IPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDN STYIVQTIKDIYAKDNTTLKKQ
FDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK
YIGNKLGSHLDVTHQFKS STKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPE
QKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELN
NIKGEPRIKKTI GKKVN SIEKLTTDVL GNVFTNTQYTKPQLLFKRGN.

[00174] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7022 (designated herein as sRGN2):
MNQKFILGLDIGIT SVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
ERVKSLLSEYKIISGLAPTNNQPYNIRVKGLTEQLTKDELAVALLHIAKRRGIHKIDVID SNDD
VGNEL STKEQLNKN SKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH
QLDENFINKYIELVEMRREYFEGP GQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYA
Y SAD LFNALNDLNNLIIQRDN SEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYR
ITKSGTPEFTEFKLYHDLKSVLFDQ SILENEDVLDQIAEILTIYQDKD SIKSKLTELDILLNEEDK
ENIAQLTGYNGTHRLSLKCIRLVLEEQWYS SRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEF
IL SPVVKRTFIQ SINVINKVIEKYGIP EDIIIELARENN SDDRKKFINNLQKKNEATRKRINEIIGQ
TGNQNAKRIVEKIRLHDQQEGKCLYSLESIALMDLLNNPQNYEVDHIIPRSVAFDNSIHNKVL
VKQIENSKKGNRTPYQYLNS SDAKLSYNQFKQHILNL SKSKDRISKKKKDYLLEERDINKFEV
QKEFINRNLVDTRYATRELT SYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRNHGY
KHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVD SEDNYSEMFIIPKQVQDIK
DFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPEKFL
MYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGS
HLDVTHQFKS STKKLVKL SIKPYRFDVYLTDKGYKFITI SYLDVLKKDNYYYIPEQKYDKLKL
GKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEPRIK
KTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00175] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7023 (designated herein as sRGN3):
MNQKFILGLDIGIT SVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
ERVKLLLTEYDLINKEQIPT SNNPYQIRVKGL SEIL SKDELAIALLHLAKRRGIHNVDVAADKE
ETASD SL STKDQINKNAKFLE SRYVCELQKERLENE GHVRGVENRFLTKDIVREAKKIIDTQM
QYYPEIDETFKEKYI SLVETRREYFEGP GQGSPFGWNGD LKKWYEMLM GHCTYFPQELRSV
KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK
GYRITKS GTPEFT SFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKD SIVAELGQLEYLM
SEADKQ SI SELT GYTGTH SL SLKCMNMIIDELWH S SMNQMEVFTYLNMRPKKYELKGYQRIP
TDMID DAIL SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK
RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS
YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER
DINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFK
KERNHGYKHHAEDALIIANADFLFKENKKLKAVN SVLEKPEIETKQLDIQVD SEDNYSEMFII
PKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF
DKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK
YIGNKLGSHLDVTHQFKS STKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPE
QKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELN
NIKGEPRIKKTI GKKVN SIEKLTTDVL GNVFTNTQYTKPQLLFKRGN.

[00176] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7024 (designated herein as sRGN3.1):
MNQKFILGLDIGIT SVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
ERVKLLLTEYDLINKEQIPT SNNPYQIRVKGL SEIL SKDELAIALLHLAKRRGIHNVDVAADKE
ETASD SL STKDQINKNAKFLE SRYVCELQKERLENE GHVRGVENRFLTKDIVREAKKIIDTQM
QYYPEIDETFKEKYI SLVETRREYFEGP GQGSPFGWNGD LKKWYEMLM GHCTYFPQELRSV
KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK
GYRITKS GTPEFT SFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKD SIVAELGQLEYLM
SEADKQ SI SELT GYTGTH SL SLKCMNMIIDELWH S SMNQMEVFTYLNMRPKKYELKGYQRIP
TDMID DAIL SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK
RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS
YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER
DINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFK
KERNHGYKHHAEDALIIANADFLFKENKKLKAVN SVLEKPEIETKQLDIQVD SEDNYSEMFII
PKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF
DKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK

YIGNKLGSHLDVTHQFKSSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIP
KDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEI
NNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[00177] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7025 (designated herein as sRGN3.2):
MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE
ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQM
QYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV
KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK
GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM
SEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP
TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK
RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS
YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER
DINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFK
KERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFII
PKQVQDIKDFRNFKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKKQ
FNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIK
LLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYI
PKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYC
EINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[00178] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7026 (designated herein as sRGN3.3):
MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE
ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQM
QYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV
KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK
GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM
SEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP
TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK
RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS
YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER
DINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFD

KYRNHGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEEDYNNVFE
TPKLVEDIKQYRDYKF SHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKK
QFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKI
KLLGNKVGNHLDVTNKYEN STKKLVKL SIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYY
IPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYC
EINNIKGEPRIKKTIGKKTE SIEKFTTDVLGNLYLHSTEKAPQLIFKRGL

[00179] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7027 (designated herein as sRGN4):
MNQKFILGLDIGIT SVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
ERVKKLLEDYNLLDQ SQIPQ STNPYAIRVKGL SEAL SKDELVIALLHIAKRRGIHNINVS SEDE
DASNEL STKEQINRNNKLLKDKYVCEVQLQRLKE GQIRGEKNRFKTTDILKEIDQLLKVQKD
YHNLDIDFINQYKEIVETRREYFE GP GKGSPYGWEGDPKAWYETLMGHCTYFPD ELRSVKY
AY SADLFNALNDLNNLVIQRD GL SKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKG
YRITKS GKPEFT SFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKD SIVAELGQLEYLMS
EADKQ SI SELT GYTGTH SL SLKCMNMIIDELWH SSMNQMEVFTYLNMRPKKYELKGYQRIPT
DMIDD AIL SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKR
INEIIGQT GNQNAKRIVEKIRLHDQQE GKCLY SLE SIPLEDLLNNPNHYEVDHIIPRSV SFDN SY
HNKVLVKQSENSKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDI
NKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKE
RNHGYKHHAEDALIIANADFLFKENKKLKAVN SVLEKPEIETKQLDIQVD SEDNYSEMFIIPK
QVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFD
KSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYI
GNKLGSHLDVTHQFKS STKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQK
YDKLKL GKAIDKNAKFIASFYKNDLIKLD GEIYKII GVN SD TRNMIELDLP DIRYKEYCELNNI
KGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00180] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7028 (designated herein as Staphylococcus hyicus Cas9 or ShyCas9):
MNNYILGLDIGITSVGYGIVD SDTREIKDAGVRLFPEANVDNNEGRRSKRGARRLKR
RRIHRLDRVKHLLAEYDLLDLTNIPKSTNPYQTRVKGLNEKL SKDELVIALLHIAKRRGIHNV
NVMMDDND SGNEL STKDQLKKNAKALSDKYVCELQLERFEQDYKVRGEKNRFKTEDFVRE
ARKLLETQSKFFEIDQTFIMRYIELIETRREYFEGPGKGSPFGWEGNIKKWFEQMMGHCTYFP
EELRSVKYSYSAELFNALNDLNNLVITRDEDAKLNYGEKFQIIENVFKQKKTPNLKQIAIEIGV
HETEIKGYRVNKSGKPEFTQFKLYHDLKNIFKDPKYLNDIQLMDNIAEIITIYQDAESIIKELNQ
LPELL SEREKEKI SAL SGYSGTHRLSLKCINLLLDDLWE S SLNQMELFTKLNLKPKKIDLSQQH
KIP SKLVDDFILSPVVKRAFIQSIQVVNAIIDKYGLPEDIIIELARENNSDDRRKFLNQLQKQNE

ETRKQVEKVLREYGNDNAKRIVQKIKLHNMQEGKCLYSLKDIPLEDLLRNPHHYEVDHIIPRS
VAFDNSMHNKVLVRADENSKKGNRTPYQYLNS SE S SLSYNEFKQHILNLSKTKDRITKKKRE
YLLEERDINKFDVQKEFINRNLVDTRYATRELTSLLKAYFSANNLDVKVKTINGSFTNYLRKV
WKFDKDRNKGYKHHAEDALHANADFLFKHNKKLRNINKVLDAP SKEVDKKRVTVQ SED EY
NQIFEDTQKAQAIKKFEIRKFSHRVDKKPNRQLINDTLY STRNIDGIEYVVESIKDIYSVNNDK
VKTKFKKDPHRLLMYRNDPQTFEKFEKVFKQYESEKNPFAKYYEETGEKIRKFSKTGQGPYI
NKIKYLRERLGRHCDVTNKYINSRNKIVQLKIYSYRFDIYQYGNNYKMITISYIDLEQKSNYY
YISREKYEQKKKDKQIDD SYKFIGSFYKNDIINYNGEMYRVIGVND SEKNKIQLDMIDISIKDY
MELNNIKKT GVIYKTIGKSTTHIEKYTTDILGNLYKAAPPKKPQLIFK.

[00181] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7029 (designated herein as Staphylococcus microfi Cas9 or Smi Cas9):
MEKDYILGLDIGI GSVGYGLIDYDTKSIIDAGVRLFPEANADNNL GRRAKRGARRLKRRRIHR
LERVKSLL SEYKII S GLAPTNNQPYNIRVKGLTEQLTKDELAVALLHIAKRRGIHNVDVAADK
EETASD SL STKDQINKNAKFLE SRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQ
MQYYPEIDETFKEKYISLVETRREYYEGPGKGSPYGWDADVKKWYQLMMGHCTYFPVEFRS
VKYAYTADLYNALNDLNNLTIARDDNPKLEYHEKYHIIENVFKQKRNPTLKQIAKEIGVNDI
NI S GYRVTKS GKPQFT SFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKD SIVAELGQLE
YLM SEADKQ SI SELTGYTGTH SL SLKCMNMIIDELWHS SMNQMEVFTYLNMRPKKYELKGY
QRIPTDMIDDAIL SPVVKRSFKQAIGVVNAIIKKYGLPKDIIIELARESNSAEKSRYLRAIQKKN
EKTRERIEAIIKEYGNENAKGLVQKIKLHDAQEGKCLYSLKDIPLEDLLRNPNNYDIDHIIPRS
VSFDD SMHNKVLVRREQNAKKNNQTPYQYLTSGYADIKYSVFKQHVLNLAENKDRMTKKK
REYLLEERNINKYDVQKEFINRNLVDTRYTTRELTTLLKTYFTINNLDVKVKTINGSFTDFLR
KRWGFKKNRDEGYKHHAEDALIIANADYLFKEHKLLKEIKDVSDLAGDERNSNVKDEDQYE
EVFGGYFKIEDIKKYKIKKFSHRVDKKPNRQLINDTIY STRVKDDKRYLINTLKNLYDKSNGD
LKERMQKDPESLLMYHHDPQTFEKLKIVMSQYENEKNPLAKYFEETGQYLTKYAKHDNGPA
IHKIKYYGNKLVEHLDITKNYHNPQNKVVQLSQKSFRFDVYQTDKGYKFISIAYLTLKNEKN
YYAISQEKYDQLKSEKKISNNAVFIGSFYT SDHEINNEKFRVIGVNSDKNNLIEVDRIDIRQKEF
IELEEEKKNNRIKVTIGRKTTNIEKFHTDIL GNMYKSKRPKAPQLVFKKG.

[00182] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7030 (designated herein as Staphylococcus pasteuri Cas9 or Spa Cas9):
MKEKYILGLDLGITSVGYGIINFETKKIIDAGVRLFPEANVDNNEGRRSKRGSRRLKRRRIHRL
ERVKLLLTEYDLINKEQIPT SNNPYQIRVKGL SEIL SKDELAIALLHLAKRRGIHNINVS SEDED
ASNEL STKEQINRNNKLLKDKYVCEVQLQRLKE GQIRGEKNRFKTTDILKEIDQLLKVQKDY
HNLDIDFINQYKEIVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYA
Y SAD LFNALNDLNNLIIQRDN SEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYR

ITKSGTPQFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEQSIKEELNKLPEILNEQDK
AEIAKLIGYNGTHRLSLKCIHLINEELWQT SRNQMEIFNYLNIKPNKVDLSEQNKIPKDMVND
FILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIG
QTGNQNAKRIVEKIRLHDQQEGKCLYSLE SIALMDLLNNPQNYEVDHIIPRSVAFDNSIHNKV
LVKQIENSKKGNRTPYQYLN S SD AKL SYNQFKQHILNL SKSKDRISKKKKDYLLEERDINKFE
VQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRNHG
YKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEEDYNNVFETPKLVED
IKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKKQFNKNPE
KFLMYQNDPKTFEKL SIIMKQY SD EKNPLAKYYEETGEYLTKY SKKNNGPIVKKIKLLGNKV
GNHLDVTNKYEN STKKLVKL SIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQ
ELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEINNIKG
EPRIKKTIGKKTE SIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[00183] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7031 (designated herein as Cas12i1):
MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISG
TANKDKISDDLLLAVNWFKVMPINSKPQGVSP SNLANLFQQYSGSEPDIQAQEYFASNFDTE
KHQWKDMRVEYERLLAELQL SRSDMHHDLKLMYKEKCIGLSL STAHYITSVMFGTGAKNN
RQTKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCD SYRKLRIRC SRKGATP SILKIVQDYEL
GTNHDDEVNVP SLIANLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSY SAMLENAL S
PIKGMTTKNCKFVLKQIDAKNDIKYENEPFGKIVEGFFD SPYFESDTNVKWVLHPHHIGE SNI
KTLWEDLNAIHSKYEEDIASLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRY
LYSRKDDIAVDKIIDGITFLSKKHKVEKQKINPVIQKYP SFNFGNNSKLLGKIISPKDKLKHNL
KCNRNQVDNYIWIEIKVLNTKTMRWEKHHYALS STRFLEEVYYPAT SENPPDALAARFRTKT
NGYEGKPAL SAEQIEQIRSAPVGLRKVKKRQMRLEAARQQNLLPRYTWGKDFNINICKRGN
NFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIEAHANGDGVIDYNDLPVKPIESGF
VTVESQVRDKSYDQLSYNGVKLLYCKPHVE SRRSFLEKYRNGTMKDNRGNNIQIDFMKDFE
AIADDET SLYYFNMKYCKLLQS SIRNHS SQAKEYREEIFELLRDGKLSVLKLS SL SNLSFVMF
KVAKSLIGTYFGHLLKKPKN SKSD VKAPPITDEDKQKADPEMFALRLALEEKRLNKVK SKKE
VIANKIVAKALELRDKYGPVLIKGENISDTTKKGKKS STNSFLMDWLARGVANKVKEMVM
MHQGLEFVEVNPNFT SHQDPFVHKNPENTFRARYSRCTP SELTEKNRKEIL SFL SDKP SKRPT
NAYYNE GAMAFLATYGLKKNDVLGVSLEKFKQIMANILHQRSEDQLLFP SRGGMFYLATYK
LDADAT SVNWNGKQFWVCNADLVAAYNVGLVDIQKDFKKK.

[00184] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7032 (designated herein as Cas12i2):

MS SAIKSYKSVLRPNERKNQLLKSTIQCLED GS AFFFKMLQGLFGGITPEIVRF STEQEKQQQD
IALWCAVNWFRPVSQD SLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGE SYYWNDCRQ
QYYDLCRELGVEVSDLTHDLEILCREKCLAVATE SNQNNSIISVLFGTGEKEDRSVKLRITKKI
LEAI SNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNL GAP STLEKFIAKDGQKEFDLKKLQ
TDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFA
KQRLEQFKEIQSLNNLLVVKKLNDFFD SEFFSGEETYTICVHHLGGKDL SKLYKAWEDDPAD
PENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQEC SAQDILAAAKYNQQLDRYKSQKANP SVL
GNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAA
GNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEIS
ATIN SKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAY SLWEVVKEGQYHKEL GCF
VRFIS SGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVE
AKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLS
TLETVKAVKGIIY SYF STALNASKNNPI SDEQRKEFDPELFALLEKLELIRTRKKKQKVERIAN
SLIQTCLENNIKFIRGEGDL STTNNATKKKAN SRSMDWLARGVFNKIRQLAPMHNITLFGCGS
LYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKL SQNLRAKNIGTGEYYHQGVKEF
L SHYELQDLEEELLKWRSDRKSNIPCWVLQNRL AEKLGNKEAVVYIPVRGGRIYFATHKVAT
GAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQS SDEENPDGSRIKLQLT S.

[00185] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7033 (designated herein as SpCas9):
MDKKYSIGLDIGTNSVGWAVITDEYKVP SKKFKVLGNTDRHSIKKNLIGALLFD SGETAEAT
RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDD SFFHRLEESFLVEEDKKHERHPIFGNIVDE
VAYHEKYPTIYHLRKKLVD STDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL
VQTYNQLFEENPINASGVDAKAIL SARL SKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF
KSNFDLAEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNL SDAILLSDILRVNTEITKA
PL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP
ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNE
KVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFL S GEQKKAIVDLLFKTNRKVTVKQLKE
DYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE
MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN
FMQLIHDD SLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ
NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVP SEEVVKK
MKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILD SRM
NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY
PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET

NGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW
DPKKYGGFD SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKE
VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED
NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVL SAYNKHRDKPIREQAENIIHLFTL
TNLGAPAAFKYFDTTIDRKRYT STKEVLDATLIHQ SIT GLYETRIDL SQLGGD.
Modified guide RNAs

[00186] In some embodiments, the guide RNA is chemically modified. A guide RNA comprising one or more modified nucleosides or nucleotides is called a "modified" guide RNA or "chemically modified" guide RNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified guide RNA is synthesized with a non-canonical nucleoside or nucleotide, is here called "modified." Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with "dephospho" linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3' or 5' cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).

[00187] Chemical modifications such as those listed above can be combined to provide modified guide RNAs comprising nucleosides and nucleotides (collectively "residues") that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase, or a modified sugar and a modified phosphodiester. In some embodiments, every base of a guide RNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of an guide RNA molecule are replaced with phosphorothioate groups. In some embodiments, modified guide RNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified guide RNAs comprise at least one modified residue at or near the 3' end of the RNA.

[00188] In some embodiments, the guide RNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in a modified guide RNA are modified nucleosides or nucleotides.

[00189]
Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds.
Accordingly, in one aspect the guide RNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases.
In some embodiments, the modified guide RNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[00190] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[00191] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

[00192] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[00193]
Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[00194] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g. replaced with a number of different "oxy" or "deoxy"
substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.

[00195] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar);
polyethyleneglycols (PEG), 0(CH2CH20).CH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 21-0-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include "locked"
nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, 0(CH2).-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can include "unlocked"
nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).

[00196] "Deoxy"
2' modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH).CH2CH2- amino (wherein amino can be, e.g., as described herein), -NHC(0)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy;
and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

[00197] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides.

[00198] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase.
Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

[00199] In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising sgRNA, one or more residues at one or both ends of the sgRNA
may be chemically modified, and/or internal nucleosides may be modified, and/or the entire sgRNA
may be chemically modified. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3 end modification.

[00200] Modifications of 2'-0-methyl are encompassed.

[00201] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2'-fluoro (2'-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability. Modifications of 2'-fluoro (2'-F) are encompassed.

[00202] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

[00203] Abasic nucleotides refer to those which lack nitrogenous bases.

[00204] Inverted bases refer to those with linkages that are inverted from the normal 5' to 3' linkage (i.e., either a 5' to 5' linkage or a 3' to 3' linkage).

[00205] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5' nucleotide via a 5' to 5' linkage, or an abasic nucleotide may be attached to the terminal 3' nucleotide via a 3' to 3' linkage. An inverted abasic nucleotide at either the terminal 5' or 3' nucleotide may also be called an inverted abasic end cap.

[00206] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus are modified. In some embodiments, the modification is a 2'-0-Me, 2'-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and/or performance.

[00207] In some embodiments, the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds.

[00208] In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 21-0-methyl (21-0-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-fluoro (21-F) modified nucleotide.
Ribonucleoprotein complex

[00209] In some embodiments, a composition is encompassed comprising: a) one or more guide RNAs comprising one or more guide sequences from Table 1A, Table 1B, or Table 5 and b) saCas9 (when combined with a gRNA comprising any one of or combination of SEQ ID Nos:
1-35, 1000-1078, and 3000-3069) or sluCas9 (when combined with a gRNA comprising any one of or combination of SEQ ID Nos: 100-225, 2000-2116, and 4000-4251), or any of the mutant Cas9 proteins disclosed herein. In some embodiments, the guide RNA together with a Cas9 is called a ribonucleoprotein complex (RNP).

[00210] In some embodiments, the disclosure provides for an RNP complex, wherein the guide RNA (e.g., any of the guide RNAs disclosed herein) binds to or is capable of binding to a target sequence in the dystrophin gene (e.g., a splice acceptor site or a splice donor site for exons 45, 51, or 53, including e.g., the Exon 51 intron-exon junction having the sequence ccagagtaacagtctgagtaggagctaaaatattagggtattgcaa (SEQ ID NO: 721), or a target sequence bound by any of the sequences disclosed in Table 1A, Table 1B, and 5), wherein the dystrophin gene comprises a PAM recognition sequence position upstream of the target sequence, and wherein the RNP cuts at a position that is 3 nucleotides upstream (-3) of the PAM in the dystrophin gene. In some embodiments, the RNP also cuts at a position that is 2 nucleotides upstream (-2), 4 nucleotides upstream (-4), 5 nucleotides upstream (-5), or 6 nucleotides upstream (-6) of the PAM in the dystrophin gene. In some embodiments, the RNP cuts at a position that is 3 nucleotides upstream (-3) and 4 nucleotides upstream (-4) of the PAM in the dystrophin gene.

[00211] In some embodiments, chimeric Cas9 (SaCas9 or SluCas9) nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas9 nuclease domain may be replaced with a domain from a different nuclease such as Fok 1. In some embodiments, a Cas9 nuclease may be a modified nuclease.

[00212] In some embodiments, the Cas9 is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.

[00213] In some embodiments, a conserved amino acid within a Cas9 protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas9 nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include Dl OA (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3): 759-771.
In some embodiments, the Cas9 nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain.
Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al.
(2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpfl (FnCpfl) sequence (UniProtKB - A0Q7Q2 (CPFl_FRATN)).
Further exemplary amino acid substitutions include DlOA and N580A (based on the S. aureus Cas9 protein). See, e.g., Friedland et al., 2015, Genome Biol., 16:257.

[00214] In some embodiments, the Cas9 lacks cleavase activity. In some embodiments, the Cas9 comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the Cas9 lacking cleavase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas9 nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014/0186958 Al; US 2015/0166980 Al.

[00215] In some embodiments, the Cas9 comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[00216] In some embodiments, the heterologous functional domain may facilitate transport of the Cas9 into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the Cas9 may be fused with 1-10 NLS(s). In some embodiments, the Cas9 may be fused with 1-5 NLS(s). In some embodiments, the Cas9 may be fused with 1-3 NLS(s). In some embodiments, the Cas9 may be fused with one NLS.
Where one NLS is used, the NLS may be attached at the N-terminus or the C-terminus of the Cas9 sequence, and may be directly fused/attached or fused/attached via a linker. It may also be inserted within the Cas9 sequence. In other embodiments, the Cas9 may be fused with more than one NLS.
In some embodiments, the Cas9 may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the Cas9 may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the Cas9 protein is fused with one or more SV40 NLSs.
In some embodiments, the SV40 NLS comprises the amino acid sequence of SEQ ID
NO: 713 (PKKKRKV). In some embodiments, the Cas9 protein (e.g., the SaCas9 or S1uCas9 protein) is fused to one or more nucleoplasmin NLSs. In some embodiments, the Cas protein is fused to one or more c-myc NLSs. In some embodiments, the Cas protein is fused to one or more ElA
NLSs. In some embodiments, the Cas protein is fused to one or more BP (bipartite) NLSs. In some embodiments, the nucleoplasmin NLS comprises the amino acid sequence of SEQ ID NO: 714 (KRPAATKKAGQAKKKK). In some embodiments, the Cas9 protein is fused with a c-Myc NLS. In some embodiments, the c-Myc NLS is encoded by the nucleic acid sequence of SEQ
ID NO: 722 (CCGGCAGCTAAGAAAAAGAAACTGGAT). In some embodiments, the Cas9 is fused to two 5V40 NLS sequences linked at the carboxy terminus. In some embodiments, the Cas9 may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the Cas9 may be fused with 3 NLSs. In some embodiments, the Cas9 may be fused with 3 NLSs, two linked at the N-terminus and one linked at the C-terminus. In some embodiments, the Cas9 may be fused with 3 NLSs, one linked at the N-terminus and two linked at the C-terminus. In some embodiments, the Cas9 may be fused with no NLS. In some embodiments, the Cas9 may be fused with one NLS. In some embodiments, the Cas9 may be fused with an NLS on the C-terminus and does not comprise an NLS fused on the N-terminus. In some embodiments, the Cas9 may be fused with an NLS on the N-terminus and does not comprise an NLS fused on the C-terminus. In some embodiments, the Cas9 protein is fused to an 5V40 NLS and to a nucleoplasmin NLS. In some embodiments, the Cas9 protein is fused to an 5V40 NLS and to a c-Myc NLS. In some embodiments, the 5V40 NLS is fused to the C-terminus of the Cas9, while the nucleoplasmin NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the 5V40 NLS is fused to the C-terminus of the Cas9, while the c-Myc NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the 5V40 NLS is fused to the N-terminus of the Cas9, while the nucleoplasmin NLS
is fused to the C-terminus of the Cas9 protein. In some embodiments, the 5V40 NLS is fused to the N-terminus of the Cas9, while the c-Myc NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, the 5V40 NLS is fused to the Cas9 protein by means of a linker. In some embodiments, the 5V40 NLS
and linker is encoded by the nucleic acid sequence of SEQ ID NO: 723 (ATGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC). In some embodiments, the nucleoplasmin NLS is fused to the Cas9 protein by means of a linker. In some embodiments, the c-Myc NLS is fused to the Cas9 protein by means of a linker. In some embodiments, an additional domain may be: a) fused to the N- or C-terminus of the Cas protein (e.g., a Cas9 protein), b) fused to the N-terminus of an NLS fused to the N-terminus of a Cas protein, or c) fused to the C-terminus of an NLS fused to the C-terminus of a Cas protein. In some embodiments, an NLS is fused to the N- and/or C-terminus of the Cas protein by means of a linker. In some embodiments, an NLS is fused to the N-terminus of an N-terminally-fused NLS on a Cas protein by means of a linker, and/or an NLS is fused to the C-terminus of a C-terminally fused NLS on a Cas protein by means of a linker. In some embodiments, the linker is GSVD (SEQ ID
NO: 550) or GSGS
(SEQ ID NO: 551). In some embodiments, the Cas protein comprises a c-Myc NLS
fused to the N-terminus of the Cas protein (or to an N-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises an 5V40 NLS
fused to the C-terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises a nucleoplasmin NLS fused to the C-terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS
fused to the N-terminus of the Cas protein, optionally by means of a linker, b) an 5V40 NLS
fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) a nucleoplasmin NLS fused to the C-terminus of the 5V40 NLS, optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N-terminus of the Cas protein, optionally by means of a linker, b) a nucleoplasmin NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) an 5V40 NLS fused to the C-terminus of the nucleoplasmin NLS, optionally by means of a linker.

[00217] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the Cas9. In some embodiments, the half-life of the Cas9 may be increased. In some embodiments, the half-life of the Cas9 may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the Cas9. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the Cas9. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence.
In some embodiments, the Cas9 may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rubl in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[00218] In some embodiments, the heterologous functional domain may be a marker domain.
Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein.

Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow 1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, 51, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[00219] In additional embodiments, the heterologous functional domain may target the Cas9 to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the Cas9 to muscle.

[00220] In further embodiments, the heterologous functional domain may be an effector domain.
When the Cas9 is directed to its target sequence, e.g., when a Cas9 is directed to a target sequence by a guide RNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain or a nuclease domain (e.g., a non-Cas nuclease domain). In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., US Pat. No. 9,023,649.

[00221] In some embodiments, any of the compositions disclosed herein comprising any of the guides and/or endonucleases disclosed herein is sterile and/or substantially pyrogen-free. In particular embodiments, any of the compositions disclosed herein comprise a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein "pharmaceutically acceptable carrier" includes any and all solvents (e.g., water), dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, including pharmaceutically acceptable cell culture media. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In some embodiments, the composition comprises a preservative to prevent the growth of microorganisms.
Determination of efficacy of guide RNAs

[00222] In some embodiments, the efficacy of a guide RNA is determined when delivered or expressed together with other components forming an RNP. In some embodiments, the guide RNA is expressed together with a SaCas9 or SluCas9. In some embodiments, the guide RNA is delivered to or expressed in a cell line that already stably expresses an SaCas9 or SluCas9.
In some embodiments the guide RNA is delivered to a cell as part of an RNP. In some embodiments, the guide RNA is delivered to a cell along with a nucleic acid (e.g., mRNA) encoding SaCas9 or SluCas9.

[00223] In some embodiments, the efficacy of particular guide RNAs is determined based on in vitro models. In some embodiments, the in vitro model is a cell line.

[00224] In some embodiments, the efficacy of particular guide RNAs is determined across multiple in vitro cell models for a guide RNA selection process. In some embodiments, a cell line comparison of data with selected guide RNAs is performed. In some embodiments, cross screening in multiple cell models is performed.

[00225] In some embodiments, the efficacy of particular guide RNAs is determined based on in vivo models. In some embodiments, the in vivo model is a rodent model. In some embodiments, the rodent model is a mouse which expresses a mutated dystrophin gene, e.g., a mdx mouse. In some embodiments, the in vivo model is a non-human primate, for example cynomolgus monkey.
Methods of Gene Editing and Treating DMD

[00226] This disclosure provides methods for gene editing and treating Duchenne Muscular Dystrophy (DMD). In some embodiments, any of the compositions described herein may be administered to a subject in need thereof for use in making a double or single strand break in any one or more of exons 43, 44, 45, 50, 51, or 53 of the dystrophin (DMD) gene. In some embodiments, pairs of guide RNAs described herein, in any of the vector configurations described herein, may be administered to a subject in need thereof to excise a portion of a DMD, thereby treating DMD. In some embodiments, any of the compositions described herein may be administered to a subject in need thereof for use in treating DMD. In some embodiments, a nucleic acid molecule comprising a first nucleic acid encoding one or more guide RNAs of Table 1A, Table 1B, or Table 5 and a second nucleic acid encoding either SaCas9 or SluCas9 (depending on the guide) is administered to a subject to treat DMD. In some embodiments, a single nucleic acid molecule (which may be a vector, including an AAV vector) comprising a first nucleic acid encoding one or more guide RNAs of Table 1A, Table 1B, or Table 5 and a second nucleic acid encoding either SaCas9 or SluCas9 (depending on the guide) is administered to a subject to treat DMD.

[00227] In some embodiments, any of the compositions described herein is administered to a subject in need thereof to treat Duchenne Muscular Dystrophy (DMD).

[00228] In some embodiments, any of the compositions described herein is administered to a subject in need thereof to induce a double strand break in any one or more of exons 43, 44, 45, 50, 51, or 53 of the dystrophin gene.

[00229] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell any one of the compositions described herein, wherein the cell comprises a mutation in the dystrophin gene that is known to be associated with DMD.

[00230] In particular, in some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: 1) a nucleic acid molecule comprising: a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 1-35, 1000-1078, 3000-3069; and 2) a Staphylococcus aureus Cas9 (SaCas9) or a nucleic acid encoding (SaCas9).

[00231] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: 1) a nucleic acid molecule comprising: a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 1-35, 1000-1078, 3000-3069; and 2) a Staphylococcus aureus Cas9 (SaCas9) or a nucleic acid encoding (SaCas9); wherein the cell comprises a mutation in the dystrophin gene that is known to be associated with DMD.

[00232] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; and 2) a nucleic acid encoding SaCas9.

[00233] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs:
10, 12, 15, 16, 20, 27, 28, 32, 33, 35, 1001, 1003, 1005, 1010, 1012, 1013, 1016, 1017, and 1018;
a nucleic acid encoding one or more spacer sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 10, 12, 15, 16, 20, 27, 28, 32, 33, 35, 1001, 1003, 1005, 1010, 1012, 1013, 1016, 1017, and 1018; or a nucleic acid encoding one or more spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 10, 12, 15, 16, 20, 27, 28, 32, 33, 35, 1001, 1003, 1005, 1010, 1012, 1013, 1016, 1017, and 1018; and 2) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9). In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 10, 12, 15, 16, 20, 27, 28, 32, 33, 35, 1001, 1003, 1005, 1010, 1012, 1013, 1016, 1017, and 1018; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of one or more spacer sequences selected from SEQ ID
NOs: 10, 12, 15, 16, 20, 27, 28, 32, 33, 35, 1001, 1003, 1005, 1010, 1012, 1013, 1016, 1017, and 1018; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 10, 12, 15, 16, 20, 27, 28, 32, 33, 35, 1001, 1003, 1005, 1010, 1012, 1013, 1016, 1017, and 1018; and 2) a nucleic acid encoding SaCas9. In some embodiments, the spacer sequence is SEQ
ID NO: 10. In some embodiments, the spacer sequence is SEQ ID NO: 12. In some embodiments, the spacer sequence is SEQ ID NO: 15. In some embodiments, the spacer sequence is SEQ ID NO: 16. In some embodiments, the spacer sequence is SEQ ID NO: 20. In some embodiments, the spacer sequence is SEQ ID NO: 27. In some embodiments, the spacer sequence is SEQ ID NO: 28. In some embodiments, the spacer sequence is SEQ ID NO: 32. In some embodiments, the spacer sequence is SEQ ID NO: 33. In some embodiments, the spacer sequence is SEQ ID NO: 35. In some embodiments, the spacer sequence is SEQ ID NO: 1001. In some embodiments, the spacer sequence is SEQ ID NO: 1003. In some embodiments, the spacer sequence is SEQ ID NO:
1005. In some embodiments, the spacer sequence is SEQ ID NO: 1010. In some embodiments, the spacer sequence is SEQ ID NO: 1012. In some embodiments, the spacer sequence is SEQ ID NO:
1013. In some embodiments, the spacer sequence is SEQ ID NO: 1016. In some embodiments, the spacer sequence is SEQ ID NO: 1017. In some embodiments, the spacer sequence is SEQ ID NO:
1018.

[00234] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs:
3022, 3023, 3028, 3029, 3030, 3031, 3038, 3039, 3052, 3053, 3054, 3055, 3062, 3063, 3064, 3065, 3068, and 3069; a nucleic acid encoding one or more spacer sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 3022, 3023, 3028, 3029, 3030, 3031, 3038, 3039, 3052, 3053, 3054, 3055, 3062, 3063, 3064, 3065, 3068, and 3069; or a nucleic acid encoding one or more spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 3022, 3023, 3028, 3029, 3030, 3031, 3038, 3039, 3052, 3053, 3054, 3055, 3062, 3063, 3064, 3065, 3068, and 3069; and 2) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9). In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 3022, 3023, 3028, 3029, 3030, 3031, 3038, 3039, 3052, 3053, 3054, 3055, 3062, 3063, 3064, 3065, 3068, and 3069; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of one or more spacer sequences selected from SEQ ID NOs: 3022, 3023, 3028, 3029, 3030, 3031, 3038, 3039, 3052, 3053, 3054, 3055, 3062, 3063, 3064, 3065, 3068, and 3069; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs:
3022, 3023, 3028, 3029, 3030, 3031, 3038, 3039, 3052, 3053, 3054, 3055, 3062, 3063, 3064, 3065, 3068, and 3069; and 2) a nucleic acid encoding SaCas9. In some embodiments, the spacer sequence is SEQ
ID NO: 3022. In some embodiments, the spacer sequence is SEQ ID NO: 3023. In some embodiments, the spacer sequence is SEQ ID NO: 3028. In some embodiments, the spacer sequence is SEQ
ID NO: 3029. In some embodiments, the spacer sequence is SEQ ID NO: 3030. In some embodiments, the spacer sequence is SEQ ID NO: 3031. In some embodiments, the spacer sequence is SEQ
ID NO: 3038. In some embodiments, the spacer sequence is SEQ ID NO: 3039. In some embodiments, the spacer sequence is SEQ ID NO: 3052. In some embodiments, the spacer sequence is SEQ
ID NO: 3053. In some embodiments, the spacer sequence is SEQ ID NO: 3054. In some embodiments, the spacer sequence is SEQ ID NO: 3055. In some embodiments, the spacer sequence is SEQ
ID NO: 3062. In some embodiments, the spacer sequence is SEQ ID NO: 3063. In some embodiments, the spacer sequence is SEQ ID NO: 3064. In some embodiments, the spacer sequence is SEQ
ID NO: 3065. In some embodiments, the spacer sequence is SEQ ID NO: 3068. In some embodiments, the spacer sequence is SEQ ID NO: 3069.

[00235] In particular, in some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: 1) a nucleic acid molecule comprising: a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; and 2) a Staphylococcus lugdunensis (SluCas9) or a nucleic acid molecule encoding SluCas9.

[00236] In particular, in some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: 1) a nucleic acid molecule comprising: a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; and 2) a Staphylococcus lugdunensis (SluCas9) or a nucleic acid molecule encoding SluCas9; wherein the cell comprises a mutation in the dystrophin gene that is known to be associated with DMD.

[00237] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs:
100-225, 2000-2116, and 4000-4251; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID
NOs: 100-225, 2000-2116, and 4000-4251; or a nucleic acid encoding one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; and 2) a nucleic acid encoding Staphylococcus lugdunensis (SluCas9).

[00238] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: 1) a nucleic acid molecule comprising: a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 131, 134, 135, 136, 139, 140, 141, 144, 145, 146, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225; a nucleic acid encoding a spacer sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of one or more spacer sequences selected from SEQ ID NOs: 131, 134, 135, 136, 139, 140, 141, 144, 145, 146, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 131, 134, 135, 136, 139, 140, 141, 144, 145, 146, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225; and 2) a Staphylococcus lugdunensis (SluCas9) or a nucleic acid molecule encoding SluCas9. In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 131, 134, 135, 136 ,139, 140, 141, 144, 145, 146, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of one or more spacer sequences selected from SEQ ID NOs: 131, 134, 135, 136 ,139, 140, 141, 144, 145, 146, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 131, 134, 135, 136 ,139, 144, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225; and 2) a nucleic acid encoding Staphylococcus lugdunensis (SluCas9). In some embodiments, the spacer sequence is SEQ ID NO: 131. In some embodiments, the spacer sequence is SEQ ID NO: 134. In some embodiments, the spacer sequence is SEQ ID NO: 135.
In some embodiments, the spacer sequence is SEQ ID NO: 136. In some embodiments, the spacer sequence is SEQ ID NO: 139. In some embodiments, the spacer sequence is SEQ ID
NO: 140. In some embodiments, the spacer sequence is SEQ ID NO: 141. In some embodiments, the spacer sequence is SEQ ID NO: 144. In some embodiments, the spacer sequence is SEQ ID
NO: 145. In some embodiments, the spacer sequence is SEQ ID NO: 146. In some embodiments, the spacer sequence is SEQ ID NO: 148. In some embodiments, the spacer sequence is SEQ ID
NO: 149. In some embodiments, the spacer sequence is SEQ ID NO: 150. In some embodiments, the spacer sequence is SEQ ID NO: 151. In some embodiments, the spacer sequence is SEQ ID
NO: 179. In some embodiments, the spacer sequence is SEQ ID NO: 184. In some embodiments, the spacer sequence is SEQ ID NO: 201. In some embodiments, the spacer sequence is SEQ ID
NO: 223. In some embodiments, the spacer sequence is SEQ ID NO: 224. In some embodiments, the spacer sequence is SEQ ID NO: 225.

[00239] In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: 1) a nucleic acid molecule comprising: a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 4062, 4063, 4068, 4069, 4070, 4071, 4072, 4073, 4078, 4079, 4088, 4089, 4096, 4097, 4098, 4099, 4100, 4101, 4102, 4103, 4158, 4159, 4168, 4169, 4202, 4203, 4220, 4221, 4246, 4247, 4248, 4249, 4250, 4251; a nucleic acid encoding a spacer sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of one or more spacer sequences selected from SEQ ID NOs: 4062, 4063, 4068, 4069, 4070, 4071, 4072, 4073, 4078, 4079, 4088, 4089, 4096, 4097, 4098, 4099, 4100, 4101, 4102, 4103, 4158, 4159, 4168, 4169, 4202, 4203, 4220, 4221, 4246, 4247, 4248, 4249, 4250, 4251; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs:
4062, 4063, 4068, 4069, 4070, 4071, 4072, 4073, 4078, 4079, 4088, 4089, 4096, 4097, 4098, 4099, 4100, 4101, 4102, 4103, 4158, 4159, 4168, 4169, 4202, 4203, 4220, 4221, 4246, 4247, 4248, 4249, 4250, 4251; and 2) a Staphylococcus lugdunensis (SluCas9) or a nucleic acid molecule encoding SluCas9. In some embodiments, a method of treating Duchenne Muscular Dystrophy (DMD) is provided, the method comprising delivering to a cell: a single nucleic acid molecule comprising: 1) a nucleic acid encoding one or more spacer sequences selected from SEQ ID NOs: 4062, 4063, 4068, 4069, 4070, 4071, 4072, 4073, 4078, 4079, 4088, 4089, 4096, 4097, 4098, 4099, 4100, 4101, 4102, 4103, 4158, 4159, 4168, 4169, 4202, 4203, 4220, 4221, 4246, 4247, 4248, 4249, 4250, 4251; a nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of one or more spacer sequences selected from SEQ ID NOs: 4062, 4063, 4068, 4069, 4070, 4071, 4072, 4073, 4078, 4079, 4088, 4089, 4096, 4097, 4098, 4099, 4100, 4101, 4102, 4103, 4158, 4159, 4168, 4169, 4202, 4203, 4220, 4221, 4246, 4247, 4248, 4249, 4250, 4251; or a nucleic acid encoding one or more spacer sequences that is at least 90% identical to any one of SEQ ID NOs: 4062, 4063, 4068, 4069, 4070, 4071, 4072, 4073, 4078, 4079, 4088, 4089, 4096, 4097, 4098, 4099, 4100, 4101, 4102, 4103, 4158, 4159, 4168, 4169, 4202, 4203, 4220, 4221, 4246, 4247, 4248, 4249, 4250, 4251;
and 2) a nucleic acid encoding Staphylococcus lugdunensis (SluCas9). In some embodiments, the spacer sequence is SEQ
ID NO: 4062. In some embodiments, the spacer sequence is SEQ ID NO: 4063. In some embodiments, the spacer sequence is SEQ ID NO: 4068. In some embodiments, the spacer sequence is SEQ ID NO: 4069. In some embodiments, the spacer sequence is SEQ ID NO:
4070. In some embodiments, the spacer sequence is SEQ ID NO: 4071. In some embodiments, the spacer sequence is SEQ ID NO: 4072. In some embodiments, the spacer sequence is SEQ ID NO:
4073. In some embodiments, the spacer sequence is SEQ ID NO: 4078. In some embodiments, the spacer sequence is SEQ ID NO: 4079. In some embodiments, the spacer sequence is SEQ ID NO:
4088. In some embodiments, the spacer sequence is SEQ ID NO: 4089. In some embodiments, the spacer sequence is SEQ ID NO: 4096. In some embodiments, the spacer sequence is SEQ ID NO:
4097. In some embodiments, the spacer sequence is SEQ ID NO: 4098. In some embodiments, the spacer sequence is SEQ ID NO: 4099. In some embodiments, the spacer sequence is SEQ ID NO:
4100. In some embodiments, the spacer sequence is SEQ ID NO: 4101. In some embodiments, the spacer sequence is SEQ ID NO: 4102. In some embodiments, the spacer sequence is SEQ ID NO:
4103. In some embodiments, the spacer sequence is SEQ ID NO: 4158. In some embodiments, the spacer sequence is SEQ ID NO: 4159. In some embodiments, the spacer sequence is SEQ ID NO:
4168. In some embodiments, the spacer sequence is SEQ ID NO: 4169. In some embodiments, the spacer sequence is SEQ ID NO: 4202. In some embodiments, the spacer sequence is SEQ ID NO:
4203. In some embodiments, the spacer sequence is SEQ ID NO: 4220. In some embodiments, the spacer sequence is SEQ ID NO: 4221. In some embodiments, the spacer sequence is SEQ ID NO:
4246. In some embodiments, the spacer sequence is SEQ ID NO: 4247. In some embodiments, the spacer sequence is SEQ ID NO: 4248. In some embodiments, the spacer sequence is SEQ ID NO:
4249. In some embodiments, the spacer sequence is SEQ ID NO: 4250. In some embodiments, the spacer sequence is SEQ ID NO: 4251.

[00240] In some embodiments, the methods comprise delivering to a cell a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ ID NO:
711. In some embodiments, the methods comprise delivering to a cell a nucleic acid molecule encoding SaCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ ID NO: 711. In some embodiments, the methods comprise delivering to a cell a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises an amino acid sequence selected from any one of SEQ ID
NOs: 715-717. In some embodiments, the methods comprise delivering to a cell a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises the amino acid of SEQ
ID NO: 715.

[00241] In some embodiments, the methods comprise delivering to a cell a nucleic acid molecule encoding SluCas9, wherein the SluCas9 comprises the amino acid sequence of SEQ ID NO:
712. In some embodiments, the methods comprise delivering to a cell a nucleic acid molecule encoding SluCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ ID NO: 712.
In some embodiments, the methods comprise delivering to a cell a nucleic acid molecule encoding SluCas9, wherein the SluCas9 comprises an amino acid sequence selected from any one of SEQ ID
NOs: 718-720.

[00242] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;
1001 and 1005;

1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; and 1018 and 16; and a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9). In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 10 and 15. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 10 and 16. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 12 and 16. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 1001 and 1005. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 1001 and 15. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 1001 and 16. In some embodiments, the first and second spacer sequence are selected from SEQ ID
NOs: 1003 and 1005. In some embodiments, the first and second spacer sequence are selected from SEQ
ID NOs: 16 and 1003. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 12 and 1010. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs:
12 and 1012. In some embodiments, the first and second spacer sequence are selected from SEQ ID
NOs: 12 and 1013. In some embodiments, the first and second spacer sequence are selected from SEQ
ID NOs: 10 and 1016. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 1017 and 16. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 1018 and 16.

[00243] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of any one of SEQ ID NOs:
and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; and 1018 and 16; and a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).

[00244] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence that are selected from spacer sequences that are at least 90%
identical to any one of SEQ ID
NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16;
1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005;
1017 and 16; and 1018 and 16; and a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).

[00245] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence selected from any one of SEQ ID NOs: 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; and 1018 and 16; and a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9), wherein the nucleic acid encoding the SaCas9 comprises the amino acid sequence of SEQ
ID NO: 715. In some embodiments, the first and second spacer sequence are selected from SEQ
ID NOs: 1001 and 1005. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs:
1001 and 15. In some embodiments, the first and second spacer sequence are selected from SEQ ID
NOs: 1001 and 16. In some embodiments, the first and second spacer sequence are selected from SEQ
ID NOs: 1003 and 1005. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 16 and 1003. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 12 and 1010. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 12 and 1012. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 12 and 1013. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 10 and 1016. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 1017 and 16. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 1018 and 16.

[00246] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of any one of SEQ ID NOs:
1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012;
12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; and 1018 and 16; and a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9), wherein the nucleic acid encoding the SaCas9 comprises the amino acid sequence of SEQ ID NO: 715.

[00247] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; and 1018 and 16; and a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9), wherein the nucleic acid encoding the SaCas9 comprises the amino acid sequence of SEQ
ID NO: 715.

[00248] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149;
144 and 150; 145 and 131; 145 and 151; and 146 and 148; and a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9). In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 148 and 134. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 149 and 135. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 150 and 135. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 131 and 136. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 151 and 136. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 139 and 131. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 139 and 151. In some embodiments, the first and second spacer sequence are selected from SEQ ID
NOs: 140 and 131. In some embodiments, the first and second spacer sequence are selected from SEQ
ID NOs: 140 and 151. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 141 and 148. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs:
144 and 149. In some embodiments, the first and second spacer sequence are selected from SEQ ID
NOs: 144 and 150. In some embodiments, the first and second spacer sequence are selected from SEQ
ID NOs: 145 and 131. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 145 and 151. In some embodiments, the first and second spacer sequence are selected from SEQ ID NOs: 146 and 148.

[00249] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of any one of SEQ ID NOs:
148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131;
139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; and 146 and 148; and a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9).

[00250] In some embodiments, methods are provided for treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: a nucleic acid encoding a pair of guide RNAs comprising: a first and second spacer sequence that are selected from spacer sequences that are at least 90%
identical to any one of SEQ ID
NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151;
140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131;
145 and 151; and 146 and 148; and a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9).

[00251] In some embodiments, methods are provided for excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: (i) a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA; and (ii) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
wherein the pair of guide RNAs and SaCas9 excise a portion of the exon. In some embodiments, the single nucleic acid molecule is delivered to the cell on a single vector. In some embodiments, the portions of the exon remaining after excision are rejoined with a one nucleotide insertion. In some embodiments, the portions of the exon remaining after excision are rejoined without a nucleotide insertion. Precise segmental deletion means that the dual cut process takes out a specific deletion. This precise deletion can lead to exon reframing (see e.g., Figure 6). In some embodiments, the size of excised portion is between 5 and 250, 5 and 225, 5 and 200, 5 and 190, 5 and 180, 5 and 170, 5 and 160,5 and 150,5 and 125,5 and 120,5 and 115,5 and 110,5 and 100,5 and 95,5 and 90,5 and 85,5 and 80, 5 and 75, 5 and 70, 5 and 65, 5 and 60, 5 and 55, 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and 25, 5 and 20, 5 and 15, and 5-10 nucleotides. In some embodiments, the size of excised portion is between 20 and 250, 20 and 225, 20 and 200, 20 and 190, 20 and 180, 20 and 170, 20 and 160,20 and 150,20 and 125,20 and 120,20 and 115,20 and 110,20 and 100,20 and 95,20 and 90, 20 and 85, 20 and 80, 20 and 75, 20 and 70, 20 and 65, 20 and 60, 20 and 55, 20 and 50, 20 and 45, 20 and 40, 20 and 35, 20 and 30, and 20 and 25 nucleotides. In some embodiments, the size of excised portion is between 50 and 250, 50 and 225, 50 and 200, 50 and 190, 50 and 180, 50 and 170, 50 and 160, 50 and 150, 50 and 125, 50 and 120, 50 and 115, 50 and 110, and 50 and 100 nucleotides. In some embodiments, the size of excised portion of the exon is between 8 and 167 nucleotides. In some embodiments, the exon is exon 45 of the dystrophin gene. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 10 and 15 (or SEQ ID Nos: 15 and 10). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 10 and 16 (or SEQ ID Nos: 16 and 10). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 12 and 16 (or SEQ ID Nos: 16 and 12). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 1001 and 1005 (or SEQ ID Nos: 1005 and 1001). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 1001 and 15 (or SEQ ID
Nos: 15 and 1001). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 1001 and 16 (or SEQ ID Nos: 16 and 1001). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs:
1003 and 1005 (or SEQ ID Nos: 1005 and 1003). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 16 and 1003 (or SEQ ID Nos: 1003 and 16). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs:
12 and 1010 (or SEQ ID Nos: 1010 and 12). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 12 and 1012 (or SEQ ID Nos:
1012 and 12). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID
NOs: 12 and 1013 (or SEQ ID Nos: 1013 and 12). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 10 and 1016 (or SEQ
ID Nos: 1016 and 10). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 1017 and 16 (or SEQ ID Nos: 16 and 1017). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 1018 and 16 (or SEQ ID Nos: 16 and 1018). In some embodiments, the SaCas9 comprises the amino acid sequence of SEQ ID NO:
715.

[00252] In some embodiments, methods are provided for excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising: (i) a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA; and (ii) a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
wherein the pair of guide RNAs and SluCas9 excise a portion of the exon. In some embodiments, the single nucleic acid molecule is delivered to the cell on a single vector. In some embodiments, the portions of the exon remaining after excision are rejoined with a one nucleotide insertion. In some embodiments, the portions of the exon remaining after excision are rejoined without a nucleotide insertion. In some embodiments, the size of excised portion of the exon is between 8 and 167 nucleotides. In some embodiments, the exon is exon 45 of the dystrophin gene. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 148 and 134 (or SEQ ID Nos:
134 and 148). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 149 and 135 (or SEQ ID Nos: 135 and 149). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs:
150 and 135 (or SEQ ID Nos: 135 and 150). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 131 and 136 (or SEQ ID Nos: 136 and 131). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs:
151 and 136 (or SEQ ID Nos: 136 and 151). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 139 and 131 (or SEQ ID Nos:
131 and 139). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID
NOs: 139 and 151 (or SEQ ID Nos: 151 and 139). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 140 and 131 (or SEQ
ID Nos: 131 and 140). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 140 and 151 (or SEQ ID Nos: 151 and 140). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 141 and 148 (or SEQ ID Nos:
148 and 141). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 144 and 149 (or SEQ ID Nos: 149 and 144). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs:
144 and 150 (or SEQ ID Nos: 150 and 144). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 145 and 131 (or SEQ ID Nos: 131 and 145). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs:
145 and 151 (or SEQ ID Nos: 151 and 145). In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence of SEQ ID NOs: 146 and 148 (or SEQ ID Nos:
148 and 146).

[00253] In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[00254] For treatment of a subject (e.g., a human), any of the compositions disclosed herein may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The compositions may be readily administered in a variety of dosage forms, such as injectable solutions. For parenteral administration in an aqueous solution, for example, the solution will generally be suitably buffered and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, and/or intraperitoneal administration.
Combination Therapy

[00255] In some embodiments, the invention comprises combination therapies comprising any of the methods or uses described herein together with an additional therapy suitable for ameliorating DMD.
Delivery of Guide RNA Compositions

[00256] The methods and uses disclosed herein may use any suitable approach for delivering the guide RNAs and compositions described herein. Exemplary delivery approaches include vectors, such as viral vectors; lipid nanoparticles; transfection; and electroporation. In some embodiments, vectors or LNPs associated with the single-vector guide RNAs/Cas9's disclosed herein are for use in preparing a medicament for treating DMD.
Where a vector is used, it may be a viral vector, such as a non-integrating viral vector. In some embodiments, viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 (see, e.g., SEQ ID NO: 81 of US 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, e.g., SEQ ID NO: 1 of US
2015/0111955, which is incorporated by reference herein in its entirety), or AAV9 vector, wherein the number following AAV
indicates the AAV serotype. In some embodiments, the AAV vector is a single-stranded AAV
(ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV).
Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, e.g., McCarty et al., Gene Ther. 2001;8:1248-54, Naso et al., BioDrugs 2017; 31:317-334, and references cited therein for detailed discussion of various AAV vectors. In some embodiments, the AAV
vector size is measured in length of nucleotides from ITR to ITR, inclusive of both ITRs. In some embodiments, the AAV
vector is less than 5 kb in size from ITR to ITR, inclusive of both ITRs. In particular embodiments, the AAV vector is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.85 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.8 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.75 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.7 kb in size from ITR to ITR, inclusive of both ITRs. In some embodiments, the vector is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the vector is between 4.4-4.85 kb in size from ITR to ITR, inclusive of both ITRs. In some embodiments, the vector is an AAV9 vector.

[00257] In some embodiments, the vector (e.g., viral vector, such as an adeno-associated viral vector) comprises a tissue-specific (e.g., muscle-specific) promoter, e.g., which is operatively linked to a sequence encoding the guide RNA. In some embodiments, the muscle-specific promoter is a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter.
In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. Muscle-specific promoters are described in detail, e.g., in US2004/0175727 Al; Wang et al., Expert Opin Drug Del/v. (2014) 11, 345-364;
Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the tissue-specific promoter is a neuron-specific promoter, such as an enolase promoter. See, e.g., Naso et al., BioDrugs 2017; 31:317-334;
Dashkoff et al., Mol Ther Methods Clin Dev. 2016;3:16081, and references cited therein for detailed discussion of tissue-specific promoters including neuron-specific promoters.

[00258] In some embodiments, in addition to guide RNA and Cas9 sequences, the vectors further comprise nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNA
and Cas9 include, but are not limited to, promoters, enhancers, and regulatory sequences. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA.

[00259] Lipid nanoparticles (LNPs) are a known means for delivery of nucleotide and protein cargo, and may be used for delivery of the guide RNAs, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.

[00260]
Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivering the single vectors disclosed herein.

[00261] In some embodiments, the invention comprises a method for delivering any one of the single vectors disclosed herein to an ex vivo cell, wherein the guide RNA is encoded by a vector, associated with an LNP, or in aqueous solution. In some embodiments, the guide RNA/LNP or guide RNA is also associated with a Cas9 or sequence encoding Cas9 (e.g., in the same vector, LNP, or solution).

[00262] In some embodiments, the disclosure provides for methods of using any of the guides, endonucleases, cells, or compositions disclosed herein in research methods.
For example, any of the guides or endonucleases disclosed herein may be used alone or in combination in experiments under various parameters (e.g., temperatures, pH, types of cells) or combined with other reagents to evaluate the activity of the guides and/or endonucleases.

[00263] Further embodiments encompassed by the disclosure are as follows.
Embodiment B 1 is a composition comprising a single nucleic acid molecule encoding one or more guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises:
a. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
b. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
c. a first nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ
ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
d. a first nucleic acid encoding one or more spacer sequences comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ
ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
e. a first nucleic acid encoding one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
f. a first nucleic acid encoding one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);

g. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10;
1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).
h. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15;
1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013;
and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16;
1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017;
and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
i. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15;
10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005;

and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005;
1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
j. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135;

150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131;

and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
k. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9); or 1. a first nucleic acid encoding a pair of guide RNAs that is at least 90% identical to a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134;
149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151;

and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (SluCas9).
Embodiment B 2 is the composition of claim 1, wherein the guide RNA is an sgRNA.
Embodiment B 3 is the composition of claim 1, wherein the guide RNA is modified.
Embodiment B 4 is the composition of claim 3, wherein the modification alters one or more 2' positions and/or phosphodiester linkages.
Embodiment B 5 is the composition of any one of claims 3-4, wherein the modification alters one or more, or all, of the first three nucleotides of the guide RNA.
Embodiment B 6 is the composition of any one of claims 3-5, wherein the modification alters one or more, or all, of the last three nucleotides of the guide RNA.
Embodiment B 7 is the composition of any one of claims 3-6, wherein the modification includes one or more of a phosphorothioate modification, a 2'-0Me modification, a 2'-0-MOE
modification, a 2'-F modification, a 2'-0-methine-4' bridge modification, a 3'-thiophosphonoacetate modification, or a 2' -deoxy modification.
Embodiment B 8 is the composition of any one of the preceding claims, wherein the composition further comprises a pharmaceutically acceptable excipient.
Embodiment B 9 is the composition of any one of the preceding claims, wherein the single nucleic acid molecule is associated with a lipid nanoparticle (LNP).
Embodiment B 10 is the composition of any one of claims 1-8, wherein the single nucleic acid molecule is a viral vector.

Embodiment B 11 is the composition of claim 10, wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector.
Embodiment B 12 is the composition of claim 10, wherein the viral vector is an adeno-associated virus (AAV) vector.
Embodiment B 13 is the composition of claim 12, wherein the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10, AAVrh74, or AAV9 vector, wherein the number following AAV indicates the AAV serotype.
Embodiment B 14 is the composition of claim 13, wherein the AAV vector is an AAV serotype 9 vector.
Embodiment B 15 is the composition of claim 13, wherein the AAV vector is an AAVrh10 vector.
Embodiment B 16 is the composition of claim 13, wherein the AAV vector is an AAVrh74 vector.
Embodiment B 17 is the composition of any one of claims 10-16, comprising a viral vector, wherein the viral vector comprises a tissue-specific promoter.
Embodiment B 18 is the composition of any one of claims 10-16, comprising a viral vector, wherein the viral vector comprises a muscle-specific promoter, optionally wherein the muscle-specific promoter is a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, an SPc5-12 promoter, or a CK8e promoter.
Embodiment B 19 is the composition of any one of claims 10-16, comprising a viral vector, wherein the viral vector comprises a U6, H1, or 7SK promoter.
Embodiment B 20 is the composition of any one of claims 1-19, comprising a nucleic acid encoding SaCas9, wherein the spacer sequence is selected from any one of SEQ
ID NOs: 12, 15, 16, 20, 27, 28, 32, 33, and 35.
Embodiment B 21 is the composition of any one of claims 1-20, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ
ID NO:
711.
Embodiment B 22 is the composition of any one of claims 1-20, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ ID NO:
711.

Embodiment B 23 is the composition of any one of claims 1-20, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 715-717.
Embodiment B 24 is the composition of any one of claims 1-19, comprising a nucleic acid encoding SluCas9, wherein the spacer sequence is selected from any one of SEQ
ID NOs:
131, 134, 135, 136 ,139, 144, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225.
Embodiment B 25 is the composition of any one of claims 1-19, or 24, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 comprises the amino acid sequence of SEQ
ID NO:
712.
Embodiment B 26 is the composition of any one of claims 1-19, or 24, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 is a variant of the amino acid sequence of SEQ ID
NO: 712.
Embodiment B 27 is the composition of any one of claims 1-19, or 24, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 718-720.
Embodiment B 28 is the composition of any one of claims 1-27 and a pharmaceutically acceptable excipient.
Embodiment B 29 is a composition comprising a guide RNA comprising any one of SEQ ID
NOs: 1-35, 1000-1078, or 3000-3069.
Embodiment B 30 is a composition comprising a guide RNA comprising any one of SEQ ID
NOs: 100-225, 2000-2116, or 4000-4251.
Embodiment B 31 is the composition of any one of claims 1-30 for use in treating Duchenne Muscular Dystrophy (DMD).
Embodiment B 32 is the composition of any one of claims 1-30 for use in making a double strand break in any one or more of exons 43, 44, 45, 50, 51, or 53 of the dystrophin gene.
Embodiment B 33 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell the composition of any one of claims 1-30.
Embodiment B 34 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:
a. a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, or 3000-3069;

b. a spacer sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, or 3000-3069; or c. a spacer sequence that is at least 90% identical to any one of SEQ ID
NOs: 1-35, 1000-1078, 3000-3069; and ii) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).
Embodiment B 35 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid molecule encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:
a. a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, or 4000-4251;
b. a spacer sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, or 4000-4251; or c. a spacer sequence that is at least 90% identical to any one of SEQ ID NOs:

225, 2000-2116, or 4000-4251; and ii) a nucleic acid molecule encoding Staphylococcus lugdunensis (SluCas9).
Embodiment B 36 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding a pair of guide RNAs comprising:
m. a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15;
and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005;
1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018;
n. a first and second spacer sequence comprising at least 17, 18, 19, 20, or contiguous nucleotides of any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;
1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10;
1005 and 1017; 16 and 1017; and 16 and 1018; or o. a first and second spacer sequence that is at least 90% identical to any one of SEQ ID
NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16;

1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016;
1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12;
1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and ii) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).
Embodiment B 37 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding a pair of guide RNAs comprising:
p. a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; and 146 and 148;
q. a first and second spacer sequence comprising at least 17, 18, 19, 20, or contiguous nucleotides of any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or r. a first and second spacer sequence that is at least 90% identical to any one of SEQ ID
NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and ii) a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9).
Embodiment B 38 is the method of any one of claims 34-37, wherein the single nucleic acid molecule is delivered to the cell on a single vector.

Embodiment B 39 is the method of any one of claims 34-38, comprising a nucleic acid molecule encoding SaCas9, wherein the spacer sequence is selected from any one of SEQ
ID NOs: 12, 15, 16, 20, 27, 28, 32, 33, and 35.
Embodiment B 40 is the method of any one of claims 34-39, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ
ID NO:
711.
Embodiment B 41 is the method of any one of claims 34-39, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ ID NO:
711.
Embodiment B 42 is the method of any one of claims 34-39, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 715-717.
Embodiment B 43 is the method of any one of claims 34-38, comprising a nucleic acid molecule encoding SluCas9, wherein the spacer sequence is selected from any one of SEQ
ID NOs:
131, 134, 135, 136 ,139, 144, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, 225.
Embodiment B 44 is the method of any one of claims 34-38, or 43, comprising a nucleic acid molecule encoding SluCas9, wherein the SluCas9 comprises the amino acid sequence of SEQ
ID NO: 712.
Embodiment B 45 is the method of any one of claims 34-38, or 43, comprising a nucleic acid molecule encoding SluCas9, wherein the SluCas9 is a variant of the amino acid sequence of SEQ ID NO: 712.
Embodiment B 46 is the method of any one of claims 34-38, or 43, comprising a nucleic acid molecule encoding SluCas9, wherein the SluCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 718-720.
Embodiment B 47 is a method of excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA
binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA;
and ii) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);

wherein the pair of guide RNAs and SaCas9 excise a portion of the exon.
Embodiment B 48 is a method of excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA
binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA;
and ii) a nucleic acid encoding a Staphylococcus lugdunensis (SluCas9);
wherein the pair of guide RNAs and SaCas9 excise a portion of the exon.
Embodiment B 49 is the method of any one of claims 47-48, wherein the single nucleic acid molecule is delivered to the cell on a single vector.
Embodiment B 50 is the method of any one of claims 47-49, wherein the portions of the exon remaining after excision are rejoined with a one nucleotide insertion.
Embodiment B 51 is the method of any one of claims 47-49, wherein the portions of the exon remaining after excision are rejoined without a nucleotide insertion.
Embodiment B 52 is the method of claim 51, wherein the size of excised portion of the exon is between 8 and 167 nucleotides.
Embodiment B 53 is the method of any one of claims 47-52, wherein the exon is exon 45.
Embodiment B 54 is the method of any one of claims 47, and 49-53, wherein the pair of guide RNA comprise a first and second spacer sequence selected from any one of SEQ
ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005;
1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001;
16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018;.
Embodiment B 55 is the method of any one of claims 48-53, wherein the pair of guide RNA
comprise a first and second spacer sequence selected from any one of SEQ ID
NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151;
146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151;
131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146;.
Embodiment B 56 is the method of any one of claims 34, 36, 38-39, 47, and 49-54, wherein the SaCas9 comprises the amino acid sequence of SEQ ID NO: 715.
Embodiment B 57 is the composition or method of any one of the preceding claims, wherein the single nucleic acid molecule is an AAV vector, wherein the vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA
scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding a SaCas9, a polyadenylation sequence, a promoter for expression of a second sgRNA in the same direction as the promoter for SaCas9, a second sgRNA guide sequence, and a second sgRNA scaffold sequence.
Embodiment B 58 is the composition or method of claim 57, wherein the promoter for expression of the nucleic acid encoding the first sgRNA guide sequence is an hU6 promoter.
Embodiment B 59 is the composition or method of any one of claims 57-58, wherein the promoter for expression of the nucleic acid encoding the second sgRNA guide sequence is an hU6 promoter.
Embodiment B 60 is the composition or method of claim 57, wherein the promoter for expression of the nucleic acid encoding the first sgRNA guide sequence is an 7SK
promoter.
Embodiment B 61 is the composition or method of any one of claims 57-58, or 60, wherein the promoter for expression of the nucleic acid encoding the second sgRNA guide sequence is an 7SK promoter.
Embodiment B 62 is the composition or method of any one of claims 57-58, or 60, wherein the promoter for expression of the nucleic acid encoding the second sgRNA guide sequence is an Him promoter.
Embodiment B 63 is the composition or method of any one of the preceding claims wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to a nucleic acid sequence encoding one or more nuclear localization sequences (NLSs).
Embodiment B 64 is the composition or method of any one of the preceding claims wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS).

Embodiment B 65 is the composition or method of any one of the preceding claims wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS).
Embodiment B 66 is the composition or method of any one of the preceding claims wherein the nucleic acid sequence encoding SaCas9 or SluCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS).
Embodiment B 67 is the composition or method of any one of claims 64-66, wherein the one or more NLSs is an SV40 NLS.
Embodiment B 68 is the composition or method of any one of claims 64-66, wherein the one or more NLSs is an c-Myc NLS.
EXAMPLES

[00264] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.
Example 1: Exemplary DMD sgRNAs

[00265] Guide RNA comprising the guide sequences shown in Table 1A and Table 1B below are prepared according to standard methods in a single guide (sgRNA) format. A
single AAV vector is prepared that expresses one or more of the guide RNAs and a SaCas9 (for guide sequences having SEQ ID NOs: 1-35, or SEQ ID NOs: 3000-3069) or SluCas9 (for guide sequences having SEQ ID
NOs: 100-225, or SEQ ID NOs: 4000-4251). See, Table 1A and Table 1B. The AAV
vector is administered to cells in vitro and to mice (e.g., mdx mice) in vivo to assess the ability of the AAV to express the guide RNA and Cas9, edit the targeted exon (see Table 1A and Table 1B), and thereby treat DMD.

[00266] In particular, the ability of in vivo single AAV-mediated delivery of gene-editing components to successfully remove the mutant genomic sequence by exon skipping in the cardiac and skeletal muscle cells of mdx mice is tested.
Table 1A: Exemplary DMD guide sequences (human-hg38.p12) Sequence ID No.
EXON aAS9 strand Guide sequence pam of Guide Sequence 9 EXON45 SACAS 9 + TCAGGCTTCCCAATTTTTCCTG TAGAAT
EXON45 SACAS 9 + TAGAATACTGGCATCTGTTTTT GAGGAT
11 EXON45 SACAS 9 + TGGCATCTGTTTTTGAGGATTG CTGAAT
12 EXON45 SACAS 9 + TT GCCGCT GCCCAAT GCCATCC TGGAGT

19 EXON51 SACAS 9 + TAGTAACCACAGGTT GT GTCAC CAGAGT
EXON51 SACAS 9 + GTT GT GTCACCAGAGTAACAGT CT GAGT
21 EXON51 SACAS 9 + T CT GAGTAG GAG C TAAAATAT T TT GGGT

EXON53 SACAS 9 + CCTTGGTTTCTGTGATTTTCTT TT GGAT
26 EXON53 SACAS 9 + TCC TTAGCT TCCAGCCATT GT G TTGAAT
27 EXON53 SACAS 9 + CTT GTACTT CATCCCACT GATT CTGAAT
28 EXON53 SACAS 9 + ACT GATTCT GAATTCT TTCAAC TAGAAT

100 EXON43 S LUCAS 9 + ATATAT GT GTTACCTACCCTT G TCGG
101 EXON43 S LUCAS 9 + ACATTTT GT TAACTTT TTCCCA TT GG
102 EXON43 S LUCAS 9 + CTTTTTCCCATTGGAAATCAAG CT GG
103 EXON43 S LUCAS 9 + TTTTTCCCATTGGAAATCAAGC TGGG
104 EXON43 S LUCAS 9 + TCCTGTAGCTTCACCCTTTCCA CAGG

117 EXON44 S LUCAS 9 + TAT TTAGCAT GTTCCCAATTCT CAGG
118 EXON44 S LUCAS 9 + AACAGAT CT GT CAAAT C GC CT G CAGG
119 EXON44 S LUCAS 9 + AATCGCCTGCAGGTAAAAGCAT AT GG

128 EXON45 S LUCAS 9 + AGACCTCCTGCCACCGCAGATT CAGG
129 EXON45 S LUCAS 9 + TCCCAATTTTTCCTGTAGAATA CTGG
130 EXON45 S LUCAS 9 + TAGAATACTGGCATCTGTTTTT GAGG
131 EXON45 S LUCAS 9 + TTTGCCGCTGCCCAATGCCATC CTGG
132 EXON45 S LUCAS 9 + GGAGTTCCTGTAAGATACCAAA AAGG

154 EXON50 S LUCAS 9 + AGAAT G G GAT C CAGTATAC T TA CAGG
155 EXON50 S LUCAS 9 + AGTATACTTACAGGCTCCAATA GT GG
156 EXON50 S LUCAS 9 + CAGGCTCCAATAGTGGTCAGTC CAGG
157 EXON50 S LUCAS 9 + CAATAGTGGTCAGTCCAGGAGC TAGG
158 EXON50 S LUCAS 9 + GTGGTCAGTCCAGGAGCTAGGT CAGG
159 EXON50 S LUCAS 9 + TTGCCCTCAGCTCTTGAAGTAA AC GG

170 EXON51 S LUCAS 9 + TGATCATCTCGTTGATATCCTC AAGG
171 EXON51 S LUCAS 9 + TT GAT CAAGCAGAGAAAGCCAG TCGG
172 EXON51 S LUCAS 9 + AGTCGGTAAGTTCTGTCCAAGC CCGG

173 EXON51 SLUCAS9 + GCCCGGTTGAAATCTGCCAGAG CAGG
174 EXON51 SLUCAS9 + CAGAGCAGGTACCTCCAACATC AAGG
175 EXON51 SLUCAS9 + GGTACCTCCAACATCAAGGAAG ATGG
176 EXON51 SLUCAS9 + CAAGGAAGATGGCATTTCTAGT TTGG
177 EXON51 SLUCAS9 + AGATGGCATTTCTAGTTTGGAG ATGG
178 EXON51 SLUCAS9 + ATGGCAGTTTCCTTAGTAACCA CAGG
179 EXON51 SLUCAS9 + GTCACCAGAGTAACAGTCTGAG TAGG
180 EXON51 SLUCAS9 + T CT GAGTAGGAGCTAAAATATT TTGG
181 EXON51 SLUCAS9 + CT GAGTAGGAGCTAAAATAT T T TGGG
182 EXON51 SLUCAS9 + AAATATTTTGGGTTTTTGCAAA AAGG

201 EXON53 SLUCAS9 + AAAGGTATCTTTGATACTAACC TTGG
202 EXON53 SLUCAS9 + CCTTGGTTTCTGTGATTTTCTT TTGG
203 EXON53 SLUCAS9 + CTTTTGGATTGCATCTACTGTA TAGG
204 EXON53 SLUCAS9 + TTTTGGATTGCATCTACTGTAT AGGG
205 EXON53 SLUCAS9 + ACCCTCCTTCCATGACTCAAGC TTGG
206 EXON53 SLUCAS9 + CTTCCATGACTCAAGCTTGGCT CTGG
207 EXON53 SLUCAS9 + ACATTTCATTCAACTGTTGCCT CCGG
208 EXON53 SLUCAS9 + TCAACTGTTGCCTCCGGTTCTG AAGG
209 EXON53 SLUCAS9 + TGAATTCTTTCAACTAGAATAA AAGG

Table 1B: Exemplary DMD guide sequences (20-nucleotides and 21-nucleotides) Sequence ID No. of Guide Sequence EXON CAS9 Strand Guide sequence 3000 EXON43 SACAS9 + AATGCTGCTGTCTTCTTGCT
3001 EXON43 SACAS9 + CAATGCTGCTGTCTTCTTGCT
1 EXON43 SACAS9 + GCAATGCTGCTGTCTTCTTGCT

3010 EXON44 SACAS9 + TTAGCATGTTCCCAATTCTC
3011 EXON44 SACAS9 + TTTAGCATGTTCCCAATTCTC
6 EXON44 SACAS9 + ATTTAGCATGTTCCCAATTCTC
3012 EXON44 SACAS9 + TCGCCTGCAGGTAAAAGCAT
3013 EXON44 SACAS9 + ATCGCCTGCAGGTAAAAGCAT
7 EXON44 SACAS9 + AATCGCCTGCAGGTAAAAGCAT

3016 EXON45 SACAS9 + AGGCTTCCCAATTTTTCCTG
3017 EXON45 SACAS9 + CAGGCTTCCCAATTTTTCCTG
9 EXON45 SACAS9 + TCAGGCTTCCCAATTTTTCCTG
3018 EXON45 SACAS9 + GAATACTGGCATCTGTTTTT
3019 EXON45 SACAS9 + AGAATACTGGCATCTGTTTTT
10 EXON45 SACAS9 + TAGAATACTGGCATCTGTTTTT
3020 EXON45 SACAS9 + GCATCTGTTTTTGAGGATTG
3021 EXON45 SACAS9 + GGCATCTGTTTTTGAGGATTG
11 EXON45 SACAS9 + TGGCATCTGTTTTTGAGGATTG
3022 EXON45 SACAS9 + GCCGCTGCCCAATGCCATCC
3023 EXON45 SACAS9 + TGCCGCTGCCCAATGCCATCC
12 EXON45 SACAS9 + TTGCCGCTGCCCAATGCCATCC

3036 EXON51 SACAS 9 + GTAACCACAGGTTGTGTCAC
3037 EXON51 SACAS 9 + AGTAAC CACAGGT T GT GT CAC
19 EXON51 SACAS 9 + TAGTAAC CACAGGT T GT GT CAC
3038 EXON51 SACAS 9 + T GT GT CAC CAGAGTAACAGT
3039 EXON51 SACAS 9 + T T GT GT CAC CAGAGTAACAGT
20 EXON51 SACAS 9 + GT T GT GT CAC CAGAGTAACAGT
3040 EXON51 SACAS 9 + TGAGTAGGAGCTAAAATATT
3041 EXON51 SACAS 9 + CTGAGTAGGAGCTAAAATATT
21 EXON51 SACAS 9 + TCTGAGTAGGAGCTAAAATATT

3048 EX0N53 SACAS 9 + TTGGTTTCTGTGATTTTCTT
3049 EX0N53 SACAS 9 + CTTGGTTTCTGTGATTTTCTT
25 EX0N53 SACAS 9 + CCTTGGTTTCTGTGATTTTCTT
3050 EX0N53 SACAS 9 + CTTAGCTTCCAGCCATTGTG
3051 EX0N53 SACAS 9 + CCTTAGCTTCCAGCCATTGTG
26 EX0N53 SACAS 9 + TCCTTAGCTTCCAGCCATTGTG
3052 EX0N53 SACAS 9 + TGTACTTCATCCCACTGATT
3053 EX0N53 SACAS 9 + TTGTACTTCATCCCACTGATT
27 EX0N53 SACAS 9 + CTTGTACTTCATCCCACTGATT
3054 EX0N53 SACAS 9 + TGATTCTGAATTCTTTCAAC
3055 EX0N53 SACAS 9 + CTGATTCTGAATTCTTTCAAC

28 EX0N53 SACAS 9 + ACTGATTCTGAATTCTTTCAAC

4000 EX0N43 SLUCAS9 + ATATGTGTTACCTACCCTTG
4001 EX0N43 SLUCAS9 + TATATGTGTTACCTACCCTTG
100 EX0N43 SLUCAS9 + ATATATGTGTTACCTACCCTTG
4002 EX0N43 SLUCAS9 + ATTTTGTTAACTTTTTCCCA
4003 EX0N43 SLUCAS9 + CATTTTGTTAACTTTTTCCCA
101 EX0N43 SLUCAS9 + ACATTTTGTTAACTTTTTCCCA
4004 EX0N43 SLUCAS9 + TTTTCCCATTGGAAATCAAG
4005 EX0N43 SLUCAS9 + TTTTTCCCATTGGAAATCAAG
102 EX0N43 SLUCAS9 + CTTTTTCCCATTGGAAATCAAG
4006 EX0N43 SLUCAS9 + TTTCCCATTGGAAATCAAGC
4007 EX0N43 SLUCAS9 + TTTTCCCATTGGAAATCAAGC
103 EX0N43 SLUCAS9 + TTTTTCCCATTGGAAATCAAGC
4008 EX0N43 SLUCAS9 + CTGTAGCTTCACCCTTTCCA
4009 EX0N43 SLUCAS9 + CCTGTAGCTTCACCCTTTCCA
104 EX0N43 SLUCAS9 + TCCTGTAGCTTCACCCTTTCCA

4034 EX0N44 SLUCAS9 + TTTAGCATGTTCCCAATTCT
4035 EX0N44 SLUCAS9 + ATTTAGCATGTTCCCAATTCT
117 EX0N44 SLUCAS9 + TATTTAGCATGTTCCCAATTCT
4036 EX0N44 SLUCAS9 + CAGATCTGTCAAATCGCCTG
4037 EX0N44 SLUCAS9 + ACAGATCTGTCAAATCGCCTG
118 EX0N44 SLUCAS9 + AACAGAT C T GT CAAAT C GCC T G
4038 EX0N44 SLUCAS9 + TCGCCTGCAGGTAAAAGCAT
4039 EX0N44 SLUCAS9 + AT C GCC T GCAGGTAAAAGCAT
119 EX0N44 SLUCAS9 + AATCGCCTGCAGGTAAAAGCAT

4056 EX0N45 SLUCAS9 + ACCTCCTGCCACCGCAGATT
4057 EX0N45 SLUCAS9 + GACCTCCTGCCACCGCAGATT
128 EX0N45 SLUCAS9 + AGACCTCCTGCCACCGCAGATT
4058 EX0N45 SLUCAS9 + CCAATTTTTCCTGTAGAATA
4059 EX0N45 SLUCAS9 + CCCAATTTTTCCTGTAGAATA
129 EX0N45 SLUCAS9 + TCCCAATTTTTCCTGTAGAATA
4060 EX0N45 SLUCAS9 + GAATACTGGCATCTGTTTTT
4061 EX0N45 SLUCAS9 + AGAATACTGGCATCTGTTTTT
130 EX0N45 SLUCAS9 + TAGAATACTGGCATCTGTTTTT
4062 EX0N45 SLUCAS9 + TGCCGCTGCCCAATGCCATC
4063 EX0N45 SLUCAS9 + TTGCCGCTGCCCAATGCCATC
131 EX0N45 SLUCAS9 + TTTGCCGCTGCCCAATGCCATC
4064 EX0N45 SLUCAS9 + AGTTCCTGTAAGATACCAAA
4065 EX0N45 SLUCAS9 + GAGTTCCTGTAAGATACCAAA
132 EX0N45 SLUCAS9 + GGAGTTCCTGTAAGATACCAAA

4108 EXON50 SLUCAS9 + AAT GGGAT CCAGTATAC T TA
4109 EXON50 SLUCAS9 + GAAT GGGAT CCAGTATAC T TA
154 EXON50 SLUCAS9 + AGAAT GGGAT CCAGTATAC T TA
4110 EXON50 SLUCAS9 + TATACTTACAGGCTCCAATA
4111 EXON50 SLUCAS9 + GTATACTTACAGGCTCCAATA
155 EXON50 SLUCAS9 + AGTATACTTACAGGCTCCAATA
4112 EXON50 SLUCAS9 + GGCTCCAATAGTGGTCAGTC
4113 EXON50 SLUCAS9 + AGGCTCCAATAGTGGTCAGTC
156 EXON50 SLUCAS9 + CAGGCTCCAATAGTGGTCAGTC
4114 EXON50 SLUCAS9 + ATAGTGGTCAGTCCAGGAGC
4115 EXON50 SLUCAS9 + AATAGTGGTCAGTCCAGGAGC
157 EXON50 SLUCAS9 + CAATAGTGGTCAGTCCAGGAGC
4116 EXON50 SLUCAS9 + GGTCAGTCCAGGAGCTAGGT
4117 EXON50 SLUCAS9 + TGGTCAGTCCAGGAGCTAGGT
158 EXON50 SLUCAS9 + GTGGTCAGTCCAGGAGCTAGGT
4118 EXON50 SLUCAS9 + GCCCTCAGCTCTTGAAGTAA
4119 EXON50 SLUCAS9 + TGCCCTCAGCTCTTGAAGTAA
159 EXON50 SLUCAS9 + TTGCCCTCAGCTCTTGAAGTAA

4140 EXON51 SLUCAS9 + ATCATCTCGTTGATATCCTC
4141 EXON51 SLUCAS9 + GATCATCTCGTTGATATCCTC
170 EXON51 SLUCAS9 + TGATCATCTCGTTGATATCCTC
4142 EXON51 SLUCAS9 + GAT CAAGCAGAGAAAGC CAG
4143 EXON51 SLUCAS9 + T GAT CAAGCAGAGAAAGC CAG
171 EXON51 SLUCAS9 + T T GAT CAAGCAGAGAAAGC CAG
4144 EXON51 SLUCAS9 + TCGGTAAGTTCTGTCCAAGC
4145 EXON51 SLUCAS9 + GTCGGTAAGTTCTGTCCAAGC
172 EXON51 SLUCAS9 + AGTCGGTAAGTTCTGTCCAAGC
4146 EXON51 SLUCAS9 + CCGGTTGAAATCTGCCAGAG
4147 EXON51 SLUCAS9 + CCCGGTTGAAATCTGCCAGAG
173 EXON51 SLUCAS9 + GCCCGGTTGAAATCTGCCAGAG
4148 EXON51 SLUCAS9 + GAGCAGGTACCTCCAACATC
4149 EXON51 SLUCAS9 + AGAGCAGGTACCTCCAACATC
174 EXON51 SLUCAS9 + CAGAGCAGGTACCTCCAACATC
4150 EXON51 SLUCAS9 + TACCTCCAACATCAAGGAAG
4151 EXON51 SLUCAS9 + GTACCTCCAACATCAAGGAAG
175 EXON51 SLUCAS9 + GGTACCTCCAACATCAAGGAAG
4152 EXON51 SLUCAS9 + AGGAAGATGGCATTTCTAGT
4153 EXON51 SLUCAS9 + AAGGAAGATGGCATTTCTAGT
176 EXON51 SLUCAS9 + CAAGGAAGATGGCATTTCTAGT
4154 EXON51 SLUCAS9 + ATGGCATTTCTAGTTTGGAG
4155 EXON51 SLUCAS9 + GATGGCATTTCTAGTTTGGAG
177 EXON51 SLUCAS9 + AGATGGCATTTCTAGTTTGGAG
4156 EXON51 SLUCAS9 + GGCAGTTTCCTTAGTAACCA
4157 EXON51 SLUCAS9 + TGGCAGTTTCCTTAGTAACCA
178 EXON51 SLUCAS9 + ATGGCAGTTTCCTTAGTAACCA
4158 EXON51 SLUCAS9 + CACCAGAGTAACAGT C T GAG
4159 EXON51 SLUCAS9 + T CACCAGAGTAACAGT C T GAG
179 EXON51 SLUCAS9 + GT CACCAGAGTAACAGT C T GAG
4160 EXON51 SLUCAS9 + TGAGTAGGAGCTAAAATATT
4161 EXON51 SLUCAS9 + CTGAGTAGGAGCTAAAATATT

180 EXON51 SLUCAS9 + TCTGAGTAGGAGCTAAAATATT
4162 EXON51 SLUCAS9 + GAGTAGGAGCTAAAATATTT
4163 EXON51 SLUCAS9 + TGAGTAGGAGCTAAAATATTT
181 EXON51 SLUCAS9 + CTGAGTAGGAGCTAAAATATTT
4164 EXON51 SLUCAS9 + ATATTTTGGGTTTTTGCAAA
4165 EXON51 SLUCAS9 + AATATTTTGGGTTTTTGCAAA
182 EXON51 SLUCAS9 + AAATATTTTGGGTTTTTGCAAA

4202 EX0N53 SLUCAS9 + AGGTATCTTTGATACTAACC
4203 EX0N53 SLUCAS9 + AAGGTATCTTTGATACTAACC
201 EX0N53 SLUCAS9 + AAAGGTATCTTTGATACTAACC
4204 EX0N53 SLUCAS9 + TTGGTTTCTGTGATTTTCTT
4205 EX0N53 SLUCAS9 + CTTGGTTTCTGTGATTTTCTT
202 EX0N53 SLUCAS9 + CCTTGGTTTCTGTGATTTTCTT
4206 EX0N53 SLUCAS9 + TTTGGATTGCATCTACTGTA
4207 EX0N53 SLUCAS9 + TTTTGGATTGCATCTACTGTA
203 EX0N53 SLUCAS9 + CTTTTGGATTGCATCTACTGTA
4208 EX0N53 SLUCAS9 + TTGGATTGCATCTACTGTAT
4209 EX0N53 SLUCAS9 + TTTGGATTGCATCTACTGTAT
204 EX0N53 SLUCAS9 + TTTTGGATTGCATCTACTGTAT
4210 EX0N53 SLUCAS9 + CCTCCTTCCATGACTCAAGC
4211 EX0N53 SLUCAS9 + CCCTCCTTCCATGACTCAAGC
205 EX0N53 SLUCAS9 + ACCCTCCTTCCATGACTCAAGC
4212 EX0N53 SLUCAS9 + TCCATGACTCAAGCTTGGCT
4213 EX0N53 SLUCAS9 + TTCCATGACTCAAGCTTGGCT
206 EX0N53 SLUCAS9 + CTTCCATGACTCAAGCTTGGCT
4214 EX0N53 SLUCAS9 + ATTTCATTCAACTGTTGCCT
4215 EX0N53 SLUCAS9 + CATTTCATTCAACTGTTGCCT
207 EX0N53 SLUCAS9 + ACATTTCATTCAACTGTTGCCT
4216 EX0N53 SLUCAS9 + AACTGTTGCCTCCGGTTCTG
4217 EX0N53 SLUCAS9 + CAACTGTTGCCTCCGGTTCTG
208 EX0N53 SLUCAS9 + TCAACTGTTGCCTCCGGTTCTG
4218 EX0N53 SLUCAS9 + AATTCTTTCAACTAGAATAA
4219 EX0N53 SLUCAS9 + GAATTCTTTCAACTAGAATAA
209 EX0N53 SLUCAS9 + TGAATTCTTTCAACTAGAATAA

Example 2: Evaluation of DMD sgRNAs A. Materials and Methods 1. sgRNA selection

[00267] A subset of sgRNAs targeting the DMD gene were selected for indel frequency and profile evaluation. The selected sgRNAs are shown in Table 2 and were prepared according to standard methods. The criteria used to select these sgRNAs included their potential to induce exon reframing and or skipping, in addition to the existence of a mouse, dog and a non-human primate (NHP) homologue counterpart. This selection included 13 sgRNAs located within exon 45, three sgRNAs located within exon 51 and ten sgRNAs located within exon 53. The number of predicted off target sites was determined for each sgRNA.

AdtommeyElmhet-MI 01245-0024-00PM' Table2.ExemplarysgRNAs r..) o SEQ ID
SEQ ID N
N
NO of NO of Ci5 Human Human Mouse Mouse CA
CA
Guide Guide Guide Human Guide Human Guide Guide Mouse Guide Mouse Mis- 0 Exon Cas9 type Strand ID Sequence Sequence PAM ID
Sequence Sequence PAM matches TTTGCCGCTGCCC
mE45SL 300 cTTGaCGCTGCC
EXON45 SluCas9 Reframe + E45SL4 131 AATGCCATC CTGG 4 CTGTCAGACAGAA
mE45SL 301 CTGgCAGAaAGg EXON45 SluCas9 Splice E455L7 134 AAAAGAGGT AGGG 7 AgAAAGAGGT AGGG 4 GCTGTCAGACAGA
mE45SL 302 GCTGgCAGAaAG
EXON45 SluCas9 Splice E455L8 135 AAAAAGAGG TAGG 8 gAgAAAGAGG TAGG 4 AACAGCTGTCAGA
mE45SL 303 AAgAGCTGgCAG
EXON45 SluCas9 Splice E455L9 136 CAGAAAAAA GAGG 9 AaAGgAgAAA GAGG 5 Reframe AATTGGGAAGCCT
mE45SL 304 AATTaGGAAGCt EXON45 SluCas9 Alt E455L12 139 GAATCTGCG GTGG 12 TGAgTCTGCG GTGG 3 P
Reframe TGTCAGAACATTG
mE45SL 305 TGTCAGAACAcT 0 w EXON45 SluCas9 Alt E455L17 144 AATGCAACT GGGG 17 GAATGCAACT GGGG 1 r TACAGGAACTCCA
mE45SL 306 TACAGGAACTCC w ..J
1¨k u, CA EXON45 SluCas9 Reframe E455L21 148 GGATGGCAT TGGG 21 AGGATGGCAT TGGG 0 w W
I., TTACAGGAACTCC
mE45SL 307 TTACAGGAACTC 0 I., EXON45 SluCas9 Reframe E455L22 149 AGGATGGCA TTGG 22 CAGGATGGCA TTGG 0 w Reframe 308 w ; GGTATCTTACAGG
mE45SL GaTggCTTACAG 0 I., EXON45 SluCas9 Splice E455L23 150 AACTCCAGG ATGG 23 Reframe ; TTTTGGTATCTTA
mE45SL TTcTGaTggCTT
EXON45 SluCas9 Splice E455L24 151 CAGGAACTC CAGG 24 GTCACCAGAGTAA
mE51SL 310 EXON51 SluCas9 Reframe + E51SL10 179 CAGTCTGAG TAGG 10 NA NA NA
CAACGAGATGATC
mE51SL 311 CAAtGAaATGAT
EXON51 SluCas9 Splice E515L15 184 ATCAAGCAG AAGG 15 CATCAAaCAG AAGG 3 AAAGGTATCTTTG
mE53SL 312 AAAGaTATgcTT IV
EXON53 SluCas9 Splice + E535L1 201 ATACTAACC TTGG 1 GAcACTAACC TTGG 4 n CCAAAAGAAAATC
mE53SL 313 CCAAAAGAAgAT
EXON53 SluCas9 Splice E535L10 210 ACAGAAACC AAGG 10 AAGTACAAGAACA
mE53SL 314 AgGTtCAAGAAC N

EXON53 SluCas9 Reframe E535L23 223 CCTTCAGAA CCGG 23 AgCTgCAGAA CAGG 4 N
1¨, AGTTGAAAGAATT
mE53SL 315 AGTTGAAAGAAT Ci5 .6.
EXON53 SluCas9 Reframe E535L24 224 CAGAATCAG TGGG 24 TCAGAtTCAG TGGG 1 .6.
cA
oe 26alcorney-ElcocketNo.01245-0024-00PU
TAGTTGAAAGAAT
mE53SL 316 cAGTTGAAAGAA
EX0N53 SluCas9 Reframe E53SL25 225 TCAGAATCA GTGG 25 TTCAGAtTCA GTGG 2 0 TTGCCGCTGCCCA TGGAG mE45Sa 317 TTGaCGCTGCCC TGGAG N

EXON45 SaCas9 Reframe + E45Sa4 12 ATGCCATCC T 4 N
GCGGCAAACTGTT TTGAA mE45Sa 318 GCGtCAAgCTGT CTGAA Ci5 EXON45 SaCas9 Reframe E45Sa7 15 GTCAGAACA T 7 CA
Reframe TTTTGGTATCTTA CAGGA mE45Sa TTcTGaTggCTT CAGGA
EXON45 SaCas9 Splice E45Sa8 16 CAGGAACTC T 8 GTTGTGTCACCAG CTGAG mE51Sa 320 EXON51 SaCas9 Reframe + E51Sa2 20 AGTAACAGT T 2 NA NA NA
CTTGTACTTCATC CTGAA mE53Sa 321 CTTGaACcTCAT CTGAA
EXON53 SaCas9 Reframe + E53Sa3 27 CCACTGATT T 3 CCCACTGAaT T 3 Reframe ACTGATTCTGAAT TAGAA mE53Sa ACTGAaTCTGAA TGGAA
EXON53 SaCas9 Splice + E53Sa4 28 TCTTTCAAC T 4 CCTTCAGAACCGG TTGAA mE53Sa 323 gCTgCAGAACaG TTGAA
EXON53 SaCas9 Reframe E53Sa8 32 AGGCAACAG T 8 GAGaCAACAG T 4 P
AGTTGAAAGAATT TGGGA mE53Sa 324 w EXON53 SaCas9 Reframe E53Sa9 33 CAGAATCAG T 9 TCAGAtTCAG T 1 r 1¨k Reframe 325 w ..J
u, CA
.6. TTTTTCCTTTTAT AAGAA
mE53Sa TTcTTatTTTTA AAGAA w EXON53 SaCas9 Splice E53Sa11 35 TCTAGTTGA T 11 TTCcAGTTGA T 4 I., ATCTTACAGGAAC
mE45Sp 403 ggCTTACAGGAA w EXON45 SpCas9 Reframe E45Sp52 400 TCCAGGA TGG 52 w CACCAGAGTAACA
mE51Sp 404 CACtAGAGTAAC 0 I., EXON51 SpCas9 Reframe + E51Sp32 401 GTCTGAG TAG 32 AGTCTGAc TGG 2 TTGAAAGAATTCA
mE53Sp 405 TTGAAAGAATTC
EXON53 SpCas9 Reframe E53Sp63 402 GAATCAG TGG 63 AGAtTCAG TGG 1 1-o n cp t., t., ,¨, .6.
,.z .6.
cA
oe 2. Transfection of HECK293FT and Neuro-2a cells

[00268] To evaluate indel frequency and profile, human HEK293FT and mouse Neuro-2a cell lines were used. HEK293FT and Neuro-2a cells were transfected in 12-well plates with 750 ng plasmid + 2.25 itL of Lipofectamine 2000. Three days after transfection, cells were trypsinized and sorted for green fluorescent protein (GFP). GFP-positive cells were sorted directly into lysis buffer, and DNA extraction was performed using the GeneJet Genomic DNA Purification Kit. PCR was then performed on the DNA using exon-specific primers that targeted the relevant cut site.
3. Amplicon deep sequencing, library preparation, and data analysis

[00269] The relevant loci for each exon were amplified by PCR and the products were used to prepare sequencing libraries using MiSeq reagent kit V3. Indel analysis was performed using CRISPResso2 10-nt quantification window. (See, e.g., Clement et al., Nat Biotechnol. 2019 Mar;
37(3):224-226). Indel profiling consisted of 5 mutually exclusive indel categories, depicted in Figure 2, and provided below:
a) NE: non-edited;
b) RF.+1: 1-nucleotide (nt) insertion leading to reframe;
c) RF.Other: indels other than 1-nt insertion leading to reframe:
= Deletion: not extending outside of the reframing window = Insertion: < 17-nt (i.e., < 6 amino acids);
d) Exon skipping: indels that disrupt the 6-nt window of the exon/intron boundaries leading to potential exon skipping (outcome requiring validation):
= The indel has >= 9-nt overlap with the splicing window (to disrupt the GT/AG
splicing sites); or 3) OE: Other indels.

[00270] The following sequences of selected primers were used for amplification of the specific human locus containing the sgRNA targeting sites (Tables 3A-3B) and the specific mouse locus containing the sgRNA targeting sites (Tables 4A-4B).
Table 3A. Primers for Amplification (Human) Targeting Primer ID Sequence exon 45 hE45-T7E1-F2 GTCTTTCTGTCTTGTATCCTTTGG (SEQ ID NO: 800) hE45-T7E1-R2 AATGTTAGTGCCTTTCACCC (SEQ ID NO: 801) 51 Ex51_MiSeq_R CATGAATAAGAGTTTGGCTCAAATTG (SEQ ID NO: 802) Ex51_MiSeq_F GAGAGTAAAGTGATTGGTGGAAAATC (SEQ ID NO: 803) hEX53 Fl AAATGTGAGATAACGTTTGGAAG (SEQ ID NO: 804) hEX53_R1 TTTCAGCTTTAACGTGATTTTCTG (SEQ ID NO: 805) Table 3B. Primer + MiSeq Adapter Sequence (Human) Exon ID Sequence MiSeq_hE45-T7E1- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTCTTTCTGTCTTGTATCCTTTG
F2 G (SEQ ID NO: 806) MiSeq_hE45-T7E1- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATGTTAGTGCCTTTCACCC
R2 (SEQ ID NO: 807) MiSeq_Ex51_MiSeq_ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATGAATAAGAGTTTGGCTCAAA
51 TTG (SEQ ID NO: 808) MiSeq_Ex51_MiSeq_ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAGAGTAAAGTGATTGGTGGAA
AATC (SEQ ID NO: 809) TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAATGTGAGATAACGTTTGGAAG
53 MiSeq_hEX53_Fl (SEQ ID NO: 810) GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCAGCTTTAACGTGATTTTC
MiSeq_hEX53_R1 TG (SEQ ID NO: 811) Table 4A. Primers for Amplification (Mouse) Targeting Primer ID Sequence exon mEx45 F3 TTTTCAGTGTAACTGCACATAAGAG (SEQ ID NO: 812) mEx45_R3 GCAAAAGTTGTCATTGTTGCT (SEQ ID NO: 813) 51 mEx51 F3 AAAATTGGCTCTTTTGCTTG (SEQ ID NO: 814) mEx5l_R3 CACAGAGAAAAGGTAGCCTAAAAA (SEQ ID NO: 815) mEx53 F3 GCACCTTGGATATATTTAATGAGAA (SEQ ID NO: 816) mEx53_R3 CAAGAATTCCACTTTTCACTTCC (SEQ ID NO: 817) Table 4B. Primer + MiSeq Adapter Sequence (Mouse) Exon ID Sequence TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTTCAGTGTAACTGCACATA
mEx45 F3 AGAG (SEQ ID NO: 818) GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAAAAGTTGTCATTGTTGC
mEx45_R3 T (SEQ ID NO: 819) TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAATTGGCTCTTTTGCTTG
51 mEx51 F3 (SEQ ID NO: 820) GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACAGAGAAAAGGTAGCCTA
mEx5l_R3 AAAA (SEQ ID NO: 821) TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCACCTTGGATATATTTAATG
mEx53 F3 AGAA (SEQ ID NO: 822) GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGAATTCCACTTTTCACT
mEx53_R3 TOO (SEQ ID NO: 823) B. Results

[00271] A set of exemplary DMD sgRNAs were evaluated for indel frequency and editing profile with either SaCas9 or SluCas9 (as indicated in Table 2). Among this selection, 13 sgRNAs were located within exon 45, 3 sgRNAs were located within exon 51 and 10 sgRNAs were located within exon 53. To evaluate indel frequency and profile, plasmid transfection was performed in HEK293FT
and Neuro-2a cell lines.

[00272] The average indel frequency of sgRNAs targeting exon 45 was determined in HEK293FT cells (Figure 3A) and in Neuro-2a cells (Figure 3B), with a high-performing SpCas9 sgRNA (E45Sp52) included as a reference. Of the exon 45-targeting sgRNAs evaluated, 11 sgRNAs showed an average total indel frequency higher than 50% in HEK293FT cells and a similar result was found for 10 sgRNAs tested in the Neuro-2a cell line. With regard to indel profile, E45Sa4 and E45SL24 were found to have the highest percent of combined indels that could lead to potential exon reframing/ skipping in HEK293FT cells. In Neuro-2a cells, mE45Sa4 and mE45SL23 showed the highest ranking with respect to these potential outcomes. The sgRNAs E45SL17 and E45SL12 were not applicable to A44 mutation.

[00273] The average indel frequency of sgRNAs targeting exon 51 was determined in HEK293FT
cells (Figure 4A) and in Neuro-2a cells (Figure 4B), with a high-performing SpCas9 sgRNA
(E51Sp32) included as a reference. Figure 4A shows an indel frequency higher than 50% and a combined indel profile that could lead to potential reframing / skipping above 25% for E51Sa2 and E51SL10 in HEK293FT cells. Due to the reduced level of sequence homology for these sgRNAs in the mouse locus, these sgRNAs could not be evaluated in the Neuro-2a cell line (e.g., sgRNAs E51Sa2 and E51SL10 do not have a mouse homolog and see Figure 4B).

[00274] The average indel frequency of sgRNAs targeting exon 53 was determined in HEK293FT
cells (Figure 5A) and in Neuro-2a cells (Figure 5B), with a high-performing SpCas9 sgRNA
(E55Sp63) included as a reference. Figure 5A shows an indel frequency higher than 50% for 4 sgRNAs within exon 53 in HEK293FT cells, and Figure 5B shows a comparable result for 2 sgRNAs in Neuro-2a cells. Of the exon 53-targeting sgRNAs evaluated, E53Sa3 and mE53SL23 show the highest percent of combined indels with potential for reframing / skipping in both cell lines.
Example 3: Exemplary DMD Guide RNAs for SaCas9 and SluCas9 Variants

[00275]
Additional exemplary DMD guide RNAs were designed that may be used with SaCas9 and variants of SaCas9 (e.g., guide sequences having SEQ ID NOs: 1000-1078) and SluCas9 and variants of SluCas9 (e.g., guide sequences having SEQ ID NOs: 2000-2116) in Table 5 below. In particular, guide RNAs were designed based on SaCas9-KKH (for guide sequences having SEQ ID
NOs: 1000-1078) with a PAM sequence NNNRRT (N is any nucleotide, R is purine) and SluCas9-KH (for guide sequences having SEQ ID NOs: 2000-2116) with a PAM sequence NNRG.

[00276] Guide RNAs were designed focusing on genomic coordinate regions within exons 45, 51, and 53. For exon 45, the design region was genomic coordinates chrX: 31968307-31968546. For exon 51, the design region was genomic coordinates chrX: 31773928-31774224.
For exon 53, the design region was genomic coordinates chrX: 31679343-31679618.

[00277] Off-target site prediction was computationally performed for two sets of mismatches: (1) 3 mismatches + 0 bulge; and (2) 2 mismatches + 1 bulge. Results are shown in Table 5.

[00278] Guide RNA comprising the guide sequences shown in Table 5 below are prepared according to standard methods in a single guide (sgRNA) format. A single AAV
vector is prepared that expresses the guide RNA and a variant SaCas9 (for guide sequences having SEQ ID NOs: 1000-1078) or the guide RNA and a variant SluCas9 (for guide sequences having SEQ
ID NOs: 2000-2116). See, Table 5. The AAV vector is administered to cells in vitro and to mice (e.g., mdx mice) in vivo to assess the ability of the AAV to express the guide RNA and Cas9, edit the targeted exon (see Table 5), and thereby treat DMD.

[00279] In particular, the ability of in vivo single AAV-mediated delivery of gene-editing components to successfully remove the mutant genomic sequence by exon skipping in the cardiac and skeletal muscle cells of mdx mice is tested.

Table5:AdditionalDMDGuideSequences(human-hg38.p12) r..) o r..) r..) Genomic Ci5 coordinate SEQ ID
Un CA
chrX stop NO

Genomic (includes Guide coordinate PAM sequenc Offtargets EXON CAS9 ID chrX_start coordinates) Strand e Guide sequence PAM _grouped SACAS9 E45SaCas 45 KKH 9KKH1 31968359 31968387 +

SACAS9 E45SaCas 45 KKH 9KKH2 31968373 31968401 +

SACAS9 E45SaCas 45 KKH 9KKH3 31968387 31968415 +

P
SACAS9 E45SaCas 45 KKH 9KKH4 31968409 31968437 +
10 TAGAATACTGGCATCTGTTTTT GAGGAT 63 w r w 1¨k SACAS9 E45SaCas ..J
u, Cln 45 KKH 9KKH5 31968417 31968445 +
11 TGGCATCTGTTTTTGAGGATTG CTGAAT 35 w I., SACAS9 E45SaCas N, w 45 KKH 9KKH6 31968432 31968460 +
1002 AGGATTGCTGAATTATTTCTTC CCCAGT 42 .
w SACAS9 E45SaCas I., 45 KKH 9KKH7 31968442 31968470 +

SACAS9 E45SaCas 45 KKH 9KKH8 31968456 31968484 +

SACAS9 E45SaCas 45 KKH 9KKH9 31968471 31968499 +

SACAS9 E45SaCas 45 KKH 9KKH10 31968484 31968512 +

IV
SACAS9 E45SaCas n 45 KKH 9KKH11 31968495 31968523 +

SACAS9 E45SaCas CP
45 KKH 9KKH12 31968517 31968545 +

N
SACAS9 E45SaCas Ci5 13 GAGGTAGGGCGACAGATCTAAT AGGAAT 10 .6.
.6.
cA
oe SACAS 9 E45S a Ca s N

SACAS 9 E45S a Ca s N

Ci5 Un SACAS 9 E45S a Ca s CA

SACAS 9 E45S a Ca s SACAS 9 E45S a Ca s SACAS 9 E45S a Ca s SACAS 9 E45S a Ca s SACAS 9 E45S a Ca s P

L.

SACAS 9 E45S a Ca s L.
I
..]
I:: \ 45 KKH 9KKH22 31968421 31968449 - 1015 AATAATTCAGCAATCCTCAAAA ACAGAT 99 u, L.
IV
SACAS 9 E45S a Ca s n, GCAACTGGGGAAGAAATAATTC AGCAAT 43 L.

LO
I
SACAS 9 E45S a Ca s CATTGAATGCAACTGGGGAAGA AATAAT 54 n, SACAS 9 E45S a Ca s SACAS 9 E45S a Ca s SACAS 9 E45S a Ca s S LUCAS E45S LCa s IV
n 45 9KH 9KH1 31968332 31968358 + 2000 S LUCAS E45S LCa s CP
45 9KH 9KH2 31968342 31968368 + 2001 N
S LUCAS E45S LCa s 45 9KH 9KH3 31968359 31968385 + 2002 TGTTTGCAGACCTCCTGCCACC GCAG 9 Ci5 .6.
.6.
cA
oe SLUCAS E45S LCa s 45 9KH 9KH4 31968365 31968391 +

N

SLUCAS E45S LCa s N
45 9KH 9KH5 31968366 31968392 +

Ci5 Un SLUCAS E45S LCa s CA

45 9KH 9KH6 31968386 31968412 +

SLUCAS E45S LCa s 45 9KH 9KH7 31968394 31968420 +

SLUCAS E45S LCa s 45 9KH 9KH8 31968408 31968434 +

SLUCAS E45S LCa s 45 9KH 9KH9 31968409 31968435 +

SLUCAS E45S LCa s 45 9KH 9KH10 31968433 31968459 +

SLUCAS E45S LCa s P
45 9KH 9KH11 31968457 31968483 +

L.

SLUCAS E45S LCa s L.
I
..]
I:: \ 45 9KH 9KH12 31968483 31968509 +
131 TTTGCCGCTGCCCAATGCCATC CTGG 2 u, L.
1¨k IV
SLUCAS E45S LCa s n, 45 9KH 9KH13 31968485 31968511 +
2008 TGCCGCTGCCCAATGCCATCCT GGAG 7 L.

LO
I
SLUCAS E45S LCa s 45 9KH 9KH14 31968495 31968521 +
2009 CAATGCCATCCTGGAGTTCCTG TAAG 14 n, SLUCAS E45S LCa s 45 9KH 9KH15 31968506 31968532 +

SLUCAS E45S LCa s 45 9KH 9KH16 31968507 31968533 +

SLUCAS E45S LCa s SLUCAS E45S LCa s IV
n SLUCAS E45S LCa s CP

N
SLUCAS E45S LCa s 134 CT GT CAGACAGAAAAAAGAG GT AG G G 28 Ci5 .6.
.6.
cA
oe SLUCAS E45S LCa s N

SLUCAS E45S LCa s N

Ci5 Un SLUCAS E45S LCa s CA

SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s P

L.

SLUCAS E45S LCa s L.
I
..]
I:: \ 45 9KH 9KH29 31968369 31968395 -137 AAGCCTGAATCTGCGGTGGCAG GAGS 11 u, L.
t`J
IV
SLUCAS E45S LCa s n, 2019 GAAGCCTGAATCTGCGGTGGCA GGAG 15 L.

LO
I
SLUCAS E45S LCa s 138 GGGAAGCCTGAATCTGCGGTGG CAGG 8 n, SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s IV
n SLUCAS E45S LCa s CP

N
SLUCAS E45S LCa s 142 T GC CAGTATT CTACAGGAAAAA TTGG 17 Ci5 .6.
.6.
cA
oe SLUCAS E45S LCa s N

SLUCAS E45S LCa s N

Ci5 Un SLUCAS E45S LCa s CA

SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s P

L.

SLUCAS E45S LCa s L.
I
..]
I:: \ 45 9KH 9KH46 31968455 31968481 -146 GTTGTCAGAACATTGAATGCAA CTGG 12 u, L.
W
IV
SLUCAS E45S LCa s n, 2027 ATTGGGCAGCGGCAAACTGTTG TCAG 3 L.

LO
I
SLUCAS E45S LCa s 147 AACTCCAGGATGGCATTGGGCA GCGG 9 n, SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s SLUCAS E45S LCa s IV
n SLUCAS E45S LCa s CP

N
SLUCAS E45S LCa s 2029 TTTTTGGTATCTTACAGGAACT CCAG 21 Ci5 .6.
.6.
cA
oe SLUCAS E45S LCa s N

SLUCAS E45S LCa s N

Ci5 Un SACAS 9 E51S a Ca s CA

51 KKH 9KKH1 31773943 31773971 + 1019 SACAS 9 E51S a Ca s 51 KKH 9KKH2 31773946 31773974 + 1020 SACAS 9 E51S a Ca s 51 KKH 9KKH3 31773958 31773986 + 1021 SACAS 9 E51S a Ca s 51 KKH 9KKH4 31773969 31773997 + 1022 SACAS 9 E51S a Ca s 51 KKH 9KKH5 31773992 31774020 + 1023 SACAS 9 E51S a Ca s P
51 KKH 9KKH6 31774005 31774033 + 1024 L.

SACAS 9 E51S a Ca s L.
I:: \ 51 KKH 9KKH7 31774023 31774051 + 1025 ATAACTT
GAT CAAG CAGAGAAA GCCAGT 101 u, L.
.6, IV
SACAS 9 E51S a Ca s n, 51 KKH 9KKH8 31774027 31774055 + 1026 CT T GAT
CAAGCAGAGAAAG C CA GT C GGT 55 L.

LO
I
SACAS 9 E51S a Ca s 51 KKH 9KKH9 31774031 31774059 + 1027 AT CAAG
CAGAGAAAGC CAGT C G GTAAGT 16 n, SACAS 9 E51S a Ca s 51 KKH 9KKH10 31774047 31774075 + 1028 SACAS 9 E51S a Ca s 51 KKH 9KKH11 31774053 31774081 + 1029 SACAS 9 E51S a Ca s 51 KKH 9KKH12 31774067 31774095 + 1030 SACAS 9 E51S a Ca s IV
n 51 KKH 9KKH13 31774088 31774116 + 1031 AG CAG

SACAS 9 E51S a Ca s CP
51 KKH 9KKH14 31774100 31774128 + 1032 CAACAT

N
SACAS 9 E51S a Ca s 51 KKH 9KKH15 31774108 31774136 + 1033 AGGAAGATGGCATTTCTAGTTT GGAGAT 62 Ci5 .6.
.6.
cA
oe SACAS 9 E51S a Ca s 51 KKH 9KKH16 31774114 31774142 +

N

SACAS 9 E51S a Ca s N
51 KKH 9KKH17 31774123 31774151 +

Ci5 Un SACAS 9 E51S a Ca s CA

51 KKH 9KKH18 31774133 31774161 +

SACAS 9 E51S a Ca s 51 KKH 9KKH19 31774147 31774175 +

SACAS 9 E51S a Ca s 51 KKH 9KKH20 31774153 31774181 +

SACAS 9 E51S a Ca s 51 KKH 9KKH21 31774159 31774187 +

SACAS 9 E51S a Ca s 51 KKH 9KKH22 31774171 31774199 +

SACAS 9 E51S a Ca s P
51 KKH 9KKH23 31774180 31774208 +

L.

SACAS 9 E51S a Ca s L.
I:: \ 51 KKH 9KKH24 31773936 31773964 -1039 AGAAGGTAT GAGAAAAAAT GAT AAAAGT 236 u, L.
Ui IV
SACAS 9 E51S a Ca s n, 1040 T CAAGCAGAAGGTAT GAGAAAA AAT GAT 99 L.

LO
I
SACAS 9 E51S a Ca s 1041 T CAT CAAG CAGAAG GTAT GAGA AAAAAT 43 n, SACAS 9 E51S a Ca s SACAS 9 E51S a Ca s SACAS 9 E51S a Ca s SACAS 9 E51S a Ca s IV
n SACAS 9 E51S a Ca s CP

N
SACAS 9 E51S a Ca s 1045 AAGT TATAAAAT CACAGAG G GT GAT G GT 77 Ci5 .6.
.6.
cA
oe SACAS 9 E51S a Ca s N

SACAS 9 E51S a Ca s N

Ci5 Un SACAS 9 E51S a Ca s CA

SACAS 9 E51S a Ca s SACAS 9 E51S a Ca s SACAS 9 E51S a Ca s SACAS 9 E51S a Ca s SACAS 9 E51S a Ca s P

L.

SACAS 9 E51S a Ca s L.
I
..]
I:: \ 51 KKH 9KKH41 31774119 31774147 - 1053 AG GAAAC
T G C CAT CT CCAAACT AGAAAT 63 u, L.
I:: \
IV
SACAS 9 E51S a Ca s n, CTGTTACTCTGGTGACACAACC TGTGGT 27 L.

LO
I
SACAS 9 E51S a Ca s TAGCTCCTACTCAGACTGTTAC TCTGGT 13 n, SLUCAS E51SLCas 51 9KH 9KH1 31773969 31773995 + 2031 SLUCAS E51SLCas 51 9KH 9KH2 31773970 31773996 + 170 SLUCAS E51SLCas 51 9KH 9KH3 31774011 31774037 + 2032 SLUCAS E51SLCas IV
n 51 9KH 9KH4 31774014 31774040 + 2033 SLUCAS E51SLCas CP
51 9KH 9KH5 31774016 31774042 + 2034 T GAT T T

N
SLUCAS E51SLCas 51 9KH 9KH6 31774020 31774046 + 2035 TTTATAACTT GAT CAAGCAGAG AAAG 7 Ci5 .6.
.6.
cA
oe 00 r-- r-- (.] -1 (.] ,r, Li-) ,r, oo c \ i Lo c \ i ,-1 c) c) oo c CD u CD cp cp cp cp cp cp cp cp CD CD
cp CD CD CD
F, CD CD ,', f', f', CD CD CD f', CD
f', U
U U U U U U f', H H H U H
U H H U U U f', U U U U f', U H
U
U U U U U U U f', U H U U U H H H U
U
U U H
U
H
U
H U H U
U
H
U
H U H H
f', U f', U
f', f', U
f', H U H
f', H
U
U U U f', H U
U
U U U U U U U U H U U U f', H
H U H U U U E H H H
H
U
U
H
U

U U H H
f' U H H H U , U U U U
U U U H H U U H H f', f', H
U
U H
H H U U U f', U U H H
U f', f', U
H H U H f', U U U U U
U H U H H f', H U U H
U U U U U U U U U H U U H U H
H U f', U U U H U U U U U U f', U
H
U H U U H H f', U f', U U H
U f', U H U U U H U f', H H U
H U U f', U U U U U U U U U
U U
H H U U U U U U f', U U U H U U
U f', U U U U U
U H U U U U
f', f', U f', U f', H f', U U H
U
U
H U U H U U U U f', U U U U
H H H f', U U f', U U U f', U U
LO ,¨i c--- co c \ i 0-) c) ,¨i 00 c \ i ,r, 00 r--- oo oo r--- oo c) ,-1 c) c) ,-1 c) c) c) ,-1 c) ,-1 c) ,-1 c) ,-1 c) + + + + + + + + + + + + + + + + +
co cr) ,r, co c) oo ,r, c) ,-1 ,r, co r---c \ i ,r, co Lo r--- co cr) cr) cr) ,-1 ,-1 ,-1 ,-1 c \ i oo oo oo c) c) c) c) c) c) c) c) c) r--- r--- r--- r--- r--- r--- r--- r--- r--- r---r--- r--- r--- r--- r--- r--- r---r--- r--- r--- r--- r--- r--- r--- r--- r--- r---r--- r--- r--- r--- r--- r--- r---oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo co c \ i oo co c \ i ,r, r--- co ,r, Li-) co c \ i ,-1 Lo co c \ i c \ i oo ,r, ,r, Lo Lo Lo Lo co co co cr) c) c) c) c) c) c) c) c) c) c) c) c) c) c) c) c) r--- r--- r--- r--- r--- r--- r--- r--- r--- r---r--- r--- r--- r--- r--- r--- r---r--- r--- r--- r--- r--- r--- r--- r--- r--- r---r--- r--- r--- r--- r--- r--- r---oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo co co co co co co co co co co co co co co co co co ro ro ro ro ro ro ro ro ro ro ro ro ro ro ro ro ro U u u u u u u u u u u u u u u u u ,-] 1-1 1-1 1-1 c) 1-1 ,-1 1-1 c \ i 1-1 cn 1-1 ,r, 1-1 Ls-) 1-1 Lc, 1-1 r--- 1-1 op 1-1 (5) 1-1 c) 1-1 ,-1 1-1 c \ i 1-1 00 CI) 00 CI) 01 CI) ,¨I CI) %-1 CI) %-1 CI) %-1 CI) %-1 CI) %-1 CI) %-1 CI) %-1 CI) %-1 CI) %-1 CI) ('J CI) ('J CI) N
C1) ('J
,¨I
LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") cn cn cn cn cn cn cn cn cn cn cn cn cn cn cn cn cn U u u u u u u u u u u u u u u u u = X X X X X X X X X X X X X X X X X

CI) 01 CI) 01 CI) 01 CI) 01 CI) 01 CI) 01 CI) 01 CI) 01 CI) 01 Cf) 01 Cf) 01 Cf) 01 Cf) 01 Cf) 01 Cf) 01 Cf) 01 Cf) 01 SLUCAS E51SLCas 51 9KH 9KH24 31774115 31774141 +

N

SLUCAS E51SLCas N
51 9KH 9KH25 31774124 31774150 +

Ci5 Un SLUCAS E51SLCas CA

51 9KH 9KH26 31774133 31774159 +

SLUCAS E51SLCas 51 9KH 9KH27 31774134 31774160 +

SLUCAS E51SLCas 51 9KH 9KH28 31774146 31774172 +

SLUCAS E51SLCas 51 9KH 9KH29 31774148 31774174 +

SLUCAS E51SLCas 51 9KH 9KH30 31774154 31774180 +

SLUCAS E51SLCas P
51 9KH 9KH31 31774160 31774186 +

L.

SLUCAS E51SLCas L.
I
..]
I:: \ 51 9KH 9KH32 31774163 31774189 +
2053 T GT CAC CAGAGTAACAGT C T GA G TAG 9 u, L.
Oe IV
SLUCAS E51SLCas n, 51 9KH 9KH33 31774164 31774190 +
179 GT CAC CAGAGTAACAGT CT GAG TAGG 14 L.

LO
I
SLUCAS E51SLCas 51 9KH 9KH34 31774166 31774192 +
2054 CACCAGAGTAACAGTCTGAGTA GGAG 11 n, SLUCAS E51SLCas 51 9KH 9KH35 31774180 31774206 +

SLUCAS E51SLCas 51 9KH 9KH36 31774181 31774207 +

SLUCAS E51SLCas 51 9KH 9KH37 31774194 31774220 +

SLUCAS E51SLCas IV
n 51 9KH 9KH38 31774195 31774221 +

SLUCAS E51SLCas CP

N
SLUCAS E51SLCas 2057 TGAGAAAAAATGATAAAAGTTG GCAG 124 Ci5 .6.
.6.
cA
oe S LUCAS E51SLCas N

S LUCAS E51SLCas N

Ci5 Un S LUCAS E51SLCas CA

S LUCAS E51SLCas S LUCAS E51SLCas S LUCAS E51SLCas S LUCAS E51SLCas S LUCAS E51SLCas P

L.

S LUCAS E51SLCas L.
I
..]
I:: \ 51 9KH 9KH49 31773988 31774014 -185 GAGGGTGATGGTGGGTGACCTT GAGG 22 u, L.
IV
S LUCAS E51SLCas n, 2064 AGAGGGTGATGGTGGGTGACCT T GAG 48 L.

LO
I
S LUCAS E51SLCas 186 ATAAAATCACAGAGGGTGATGG TGGG 27 n, S LUCAS E51SLCas S LUCAS E51SLCas S LUCAS E51SLCas S LUCAS E51SLCas IV
n S LUCAS E51SLCas CP

N
S LUCAS E51SLCas 2066 TGCTTGATCAAGTTATAAAATC ACAG 11 Ci5 .6.
.6.
cA
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CI) 01 Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) Cl) ,¨I 00 00 00 00 00 00 00 00 00 00 00 00 LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") LS") SACAS9 E53S aCas N

SACAS9 E53S aCas N

Ci5 Un SACAS9 E53S aCas CA

SACAS9 E53S aCas SACAS9 E53S aCas SACAS9 E53S aCas SACAS9 E53S aCas SACAS9 E53S aCas P

L.

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31 GCAACAGTT GAAT GAAAT G T TA AAG GAT 155 u, L.
t`J
IV
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LO
I
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n SACAS9 E53S aCas CP

N
SACAS9 E53S aCas 35 TTTTTCCTTTTAT TCTAGT T GA AAGAAT 475 Ci5 .6.
.6.
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SLUCAS E53S LCa s N
53 9KH 9KH1 31679353 31679379 +

Ci5 Un SLUCAS E53S LCa s CA

53 9KH 9KH2 31679373 31679399 +

SLUCAS E53S LCa s 53 9KH 9KH3 31679391 31679417 +

SLUCAS E53S LCa s 53 9KH 9KH4 31679392 31679418 +

SLUCAS E53S LCa s 53 9KH 9KH5 31679393 31679419 +

SLUCAS E53S LCa s 53 9KH 9KH6 31679414 31679440 +

SLUCAS E53S LCa s P
53 9KH 9KH7 31679419 31679445 +

L.

SLUCAS E53S LCa s L.
---1 53 9KH 9KH8 31679425 31679451 +
206 CTTCCATGACTCAAGCTTGGCT CTGG 10 u, L.
W
IV
SLUCAS E53S LCa s n, 53 9KH 9KH9 31679436 31679462 +
2077 CAAGCTTGGCTCTGGCCTGTCC TAAG 16 L.

LO
I
SLUCAS E53S LCa s 53 9KH 9KH10 31679446 31679472 +
2078 TCTGGCCTGTCCTAAGACCTGC TCAG 18 n, SLUCAS E53S LCa s 53 9KH 9KH11 31679458 31679484 +

SLUCAS E53S LCa s 53 9KH 9KH12 31679465 31679491 +

SLUCAS E53S LCa s 53 9KH 9KH13 31679511 31679537 +

SLUCAS E53S LCa s IV
n 53 9KH 9KH14 31679519 31679545 +

SLUCAS E53S LCa s CP
53 9KH 9KH15 31679520 31679546 +

N
SLUCAS E53S LCa s 53 9KH 9KH16 31679564 31679590 +
2082 CACTGATTCTGAATTCTTT CAA CTAG 32 Ci5 .6.
.6.
cA
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N

S LUCAS E53S LCa s N
53 9KH 9KH18 31679573 31679599 +

Ci5 Un S LUCAS E53S LCa s CA

53 9KH 9KH19 31679589 31679615 +

S LUCAS E53S LCa s 53 9KH 9KH20 31679592 31679618 +

S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s P

L.

S LUCAS E53S LCa s L.

2089 AGAT GCAAT CCAAAAGAAAAT C ACAG 89 u, L.
.6, IV
S LUCAS E53S LCa s n, 2090 CCTATACAGTAGATGCAAT C CA AAAG 4 L.

LO
I
S LUCAS E53S LCa s 2091 ATGGAAGGAGGGTCCCTATACA GTAG 8 n, S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s IV
n S LUCAS E53S LCa s CP

N
S LUCAS E53S LCa s 2094 CCAGAGC CAAGCT T GAGT CAT G GAAG 18 Ci5 .6.
.6.
cA
oe SLUCAS E53S LCa s N

SLUCAS E53S LCa s N

Ci5 Un SLUCAS E53S LCa s CA

SLUCAS E53S LCa s SLUCAS E53S LCa s SLUCAS E53S LCa s SLUCAS E53S LCa s SLUCAS E53S LCa s P

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2100 TAAGGAAGAAGCT GAG CAG GT C T TAG 25 u, L.
Ui IV
SLUCAS E53S LCa s n, 217 GGAAGCTAAGGAAGAAGCT GAG CAGG 70 L.

LO
I
SLUCAS E53S LCa s 2101 T GGAAGC TAAGGAAGAAGC T GA GCAG 52 n, SLUCAS E53S LCa s SLUCAS E53S LCa s SLUCAS E53S LCa s SLUCAS E53S LCa s IV
n SLUCAS E53S LCa s CP

N
SLUCAS E53S LCa s 2016 AAAGGATTCAACACAATGGCTG GAAG 12 Ci5 .6.
.6.
cA
oe S LUCAS E53S LCa s N

S LUCAS E53S LCa s N

Ci5 Un S LUCAS E53S LCa s CA

S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s P

L.

S LUCAS E53S LCa s L.
I
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2110 G G GAT GAAGTACAAGAACACCT T CAG 18 u, L.
I:: \
IV
S LUCAS E53S LCa s n, 2111 TCAGAAT CAGT G G GAT GAAGTA CAAG 27 L.

LO
I
S LUCAS E53S LCa s 2112 AAGAATT CAGAAT CAGT G G GAT GAAG 27 n, S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s S LUCAS E53S LCa s IV
n S LUCAS E53S LCa s CP

N
S LUCAS E53S LCa s 2116 TATATTTATTTTTCCTTTTATT CTAG 978 Ci5 .6.
.6.
cA
oe Example 4: Evaluation of sgRNA Pairs A. Materials and Methods 1. sgRNA selection

[00280] A subset of SaCas9-KKH or SluCas9 sgRNAs found within the DMD gene was selected for indel frequency and profile evaluation. The criteria used to select these sgRNAs included their potential to induce exon reframing and or skipping as a pair, in addition to the existence of a mouse, dog and NHP homologue counterpart. This selection included 27 sgRNAs located within exon 45, 39 sgRNAs located within exon 51 and 29 sgRNAs located within exon 53. The number of predicted off target sites was determined for each sgRNA.
2. Transfection of HEK293FT cells

[00281] 293FT
cells were transfected in 12-well plates with 750 ng plasmid + 2.25 uL of Lipofectamine 2000. Three days after transfection, cells were trypsinized and sorted for GFP. GFP-positive cells were sorted directly into lysis buffer, and DNA extraction was performed using the Promega Maxwell RSC Blood DNA Kit. PCR was then performed on the DNA using exon-specific primers that targeted the relevant cut site.
3. Amplicon deep sequencing library preparation and data analysis

[00282] The relevant loci for each exon were amplified by PCR and the products were used to prepare sequencing libraries using MiSeq reagent kit V3 600 cycle. Indel analysis was performed using CRISPResso2 10-nt quantification window. Indel profiling consisted of 8 indel categories listed in Table 6.
Table 6.
Indel group Ranking order Definition Wild-type amplicon NE 1 insertion = 0 & deletion = 0 RF +1 2 1-nt insertion at a reframe guide RF Other 3 3n+1 deletion within the exon boundary at the only reframe guide no edits at the exon guide aggregated 3n+1 deletion within the exon boundary at the two reframe guides < 17-nt insertion at the only reframe guide no edits at the exon guide < 17-nt aggregated 3n+1 insertion at the two reframe guides Exon skipping 4 indels overlap with the splice acceptor (AG) (5'-end of the target exon) deletion with > 9-nt overlap with the splicing window at one of the two guides insertion at the exact GT/AG splicing sites and with length > 9-nt at one of the two guides OE 5 Other indels Deletion arnplicon Precise deletion 1 segmental deletion between the two cut sites (Rest) 2 SingleCut indel profiling with 3n phase Inversion amplicon OE 1 all indels 4. AAV configurations for the dual cut single vector candidates

[00283] A
combination of promoter orientations, promoter configurations, NLSs and scaffolds were selected for generating AAV plasmids and evaluation on sgRNA transgene expression, AAV
manufacturability, and editing efficiency in vitro and in vivo. AAV plasmid configurations listed in Table 7. Promoter, NLS and scaffold sequences listed in Table 8.
Table 7:
Pol III Orientation Configuration NLS1 Endonuclease NLS2 NLS3 Scaffold Promoter = 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-GSGS Nucleoplasmin- V5 hU6c GSVD GSGS
=> 4 hU6c-Cas9- c-Myc- SaCas9-KKH SV-40-GSGS Nucleoplasmin- V2 hU6c GSVD GSGS
hU6c:hU6c = 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-MCS- Nucleoplasmin- V5 hU6c GSVD GSGS GSGS
=> 4 hU6c-Cas9- c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V2 hU6c GSVD GSGS GSGS
=> 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 => 4 hU6c-Cas9- c-Myc- SaCas9-KKH SV-40-GSGS Nucleoplasmin- V2 h 6:7SK2 7SK2 GSVD GSGS
U
= 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-MCS- Nucleoplasmin- V5 => 4 hU6c-Cas9- c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V2 => 4 hU6c-Cas9-Him c-Myc- S1uCas9 SV-40-GSGS Nucleoplasmin-GSVD GSGS
hU6c:Hlm => 4 hU6c-Cas9-Him c-Myc- SaCas9-KKH SV-40-GSGS Nucleoplasmin- V2 GSVD GSGS
= 4 7SK2-Cas9- SV-40-GS S1uCas9 Nucleoplasmin N/A V5 Him = 4 7SK2-Cas9- SV-40-GS SaCas9-KKH Nucleoplasmin N/A V2 Him = 4 7SK2-Cas9- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 Him 7SK2:Hlm = 4 7SK2-Cas9- SV-40 (+) SaCas9-KKH
Nucleoplasmin N/A V2 Him = 4 7SK2-Cas9- c-Myc- S1uCas9 SV-40-MCS- Nucleoplasmin- V5 Him GSVD GSGS GSGS
= 4 7SK2-Cas9- c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V2 Him GSVD GSGS GSGS
= 4 7SK2-Cas9- c-Myc- S1uCas9 SV-40-MCS- Nucleoplasmin- V2 Him GSVD GSGS GSGS
= 4 Him-Cas9-M11 c-Myc- S1uCas9 SV-40-MCS- Nucleoplasmin- V5 Hlm:M11 GSVD GSGS GSGS
= 4 Him-Cas9-M11 c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V5 GSVD GSGS GSGS
Table 8:
Promoter Sequences GAGGGCCTATTTCCCAT GATT CCTT CATATTT GCATATACGATACAAGGCT GTTAGAGAGATA
AT T GGAATTAATT T GACT GTAAACACAAAGATAT TAGTACAAAATAC GT GACGTAGAAAGTAA
hU6c TAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACC
GTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC
(SEQ ID NO: 705) CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGA
AATCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTT

TAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTACCTC (SEQ ID
NO: 706) AATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTG
Him GGAATCTTATAAGTTCTGTATGAGACCACTCTTTCCC (SEQ ID NO: 707) ATATTTAGCATGTCGCTATGTGTTCTGGGAAACTTGACCTAAGTGTAAAGTTGAGATTTCCTT
A411 CAGGTTTATATAGTTCTGTATGAGACCACTCTTTCCC (SEQ ID NO: 708) NLS-linker AA Sequences c-Myc-GSVD PAAKKKKLDGSVD (SEQ ID NO: 36) SV-40-GS PKKKRKVGS (SEQ ID NO: 37) SV-40 (+) PKKKRKVGIHGVPAA (SEQ ID NO: 38) SV-40-GSGS GSGSPKKKRKV (SEQ ID NO: 39) SV-40-MCS- TGGGPGGGAAAGSGSPKKKRKV (SEQ ID NO: 40) GSGS
Nucleoplasmin- GSGSKRPAATKKAGQAKKKK (SEQ ID NO: 41) GSGS
Scaffold Sequences S1uCas9 scaffold GUUU CAGUACU CU GGAAACAGAAU CUACU GAAACAAGACAAUAU GU C GU
GUUUAU C C CAU CAA
V5 UUUAUUGGUGGGAU (SEQ ID NO: 922) SluCas9 scaffold GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAATGCCGTGTTT
V2 ATCTCGTCAACTTGTTGGCGAGA (SEQ ID NO: 500)

[00284]
Sequences of selected primers used for amplification of the specific human locus containing the sgRNA sites are shown in Table 9.
Table 9:
Name Sequence Mi5eq_hE45_ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTCTTTCTGTCTTGTAT
CCTTTGG (SEQ ID NO: 724) Mi5eq_hE45_ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATGTTAGTGCCTTTC
ACCC (SEQ ID NO: 725) TIDE_hE45_ F* GTCTTTCTGTCTTGTATCCTTTGG (SEQ ID NO: 726) *TIDE_hE45_F is used for Sanger sequencing.
5. Results

[00285] A set of sgRNAs found within the DMD gene was selected for evaluation of indel frequency and profile. Among this selection, 27 sgRNAs were located within exon 45, 39 sgRNAs were located within exon 51 and 29 sgRNAs were located within exon 53. To evaluate indel frequency and editing profiles, plasmid transfection was performed in HEK293FT
(Figure 6B) and Neuro-2a cell lines (Figure 6D). The average indel frequency of sgRNAs targeting exon 45 was determined in HEK293FT cells (Figure 6B) and in Neuro-2a cells (Figure 6D), with a high-performing SpCas9 sgRNA (E45Sp52) included as a reference. Of these, 20 sgRNAs pairs showed an average total indel frequency higher than 60% and 9 sgRNA pairs showed an average precise segmental deletion frequency higher than 50% in 293FT cells. Five sgRNAs tested in the Neuro-2a cell line showed an average precise segmental deletion frequency higher than 40% (Figure 6D). With regard to indel profile, sgRNA pairs E45SaKKH10/20, E45S1u21/7 and E45S1u18/4 were found to have the highest percent of precise segmental deletion that could lead to potential exon reframing in 293FT cells (Figure 6B), and sgRNA pairs E45SaKKH10/23, E45S1u17/22, E45S1u17/23 and E45S1u19/21 were found to have the highest percent of precise segmental deletion that could lead to potential exon reframing in Neuro-2a cells (Figure 6D). Table 10 shows the SEQ
ID NOs corresponding to the sgRNA identifiers in Figures 6B and 6C.
Table 10:
sgRNA identifier in certain SEQ ID NO(s) Figures, including Fig. 6 and Fig. 8 SaCas9: 2 and 7 10 and 15 SaCas9: 2 and 8 10 and 16 SaCas9: 4 and 8 12 and 16 SaCas9-KKH: 2 and 9 1001 and 1005 SaCas9-KKH: 2 and 26 1001 and 15 SaCas9-KKH: 2 and 27 1001 and 16 SaCas9-KKH: 7 and 9 1003 and 1005 SaCas9-KKH: 27 and 7 16 and 1003 SaCas9-KKH: 10 and 16 12 and 1010 SaCas9-KKH: 10 and 18 12 and 1012 SaCas9-KKH: 10 and 20 12 and 1013 SaCas9-KKH: 10 and 23 10 and 1016 SaCas9-KKH: 24 and 9 1017 and 1005 SaCas9-KKH: 24 and 27 1017 and 16 SaCas9-KKH: 25 and 27 1018 and 16 SluCas9: 21 and 7 148 and 134 SluCas9: 22 and 8 149 and 135 S1uCas9: 23 and 8 150 and 135 S1uCas9: 4 and 9 131 and 136 SluCas9: 24 and 9 151 and 136 SluCas9: 12 and 4 139 and 131 SluCas9: 12 and 24 139 and 151 SluCas9: 13 and 4 140 and 131 SluCas9: 13 and 24 140 and 151 SluCas9: 14 and 21 141 and 148 SluCas9: 17 and 22 144 and 149 SluCas9: 17 and 23 144 and 150 SluCas9: 18 and 4 145 and 131 SluCas9: 18 and 24 145 and 151 SluCas9: 19 and 21 146 and 148 SaCas9-4 12 SluCas9-24 151 EX-145 SpCas9 control (ATCTTACAGGAACTCCAGGA) (SEQ
ID NO: 727) Example 5: Testing of sgRNA Scaffold Sequences 1. Materials and Methods

[00286] Primary human skeletal muscle myoblasts (HsMM; Lonza CC-2580: lot# 20TL070666, PO) were recovered and passaged in SkBM0-2 Skeletal Muscle Myoblast Basal Medium plus SkGM0-2 SingleQuots (CC-3246, CC-3244; Lonza) in the incubator at 37 C with 5%
CO2. When HsMM culture reached approximately 80% to 90% confluence and were actively proliferating, the cells were harvested for SluCas9 ribonucleoprotein (RNP) delivery. After thawing, the cells were passaged once before SluCas9 RNP delivery.

[00287] To form SluCas9 RNPs, the appropriate amount of synthetic sgRNA (Synthego: SO#
7292552) and recombinant SluCas9 protein (Aldevron: Lot# M22536-01) were mixed in supplemented P5 Primary Cell nucleofection solution (Lonza V4XP-5032). In total, three sgRNA:SluCas9 doses were tested, including a low dose with 37.5pmo1:6.25pmo1, a middle dose 75pmo1:12.5pmol, and a high dose 150:25. The sgRNAs and SluCas9 proteins were incubated for at least 10 minutes at room temperature for Cas9-sgRNA RNP formation.

[00288] While the SluCas9 RNP was forming, HsMMs were rinsed with HEPES
buffered saline solution, dissociated from tissue culture flasks by trypsin, and centrifuge at 90xg for 10 minutes. The cell pellets were resuspended in fresh, pre-warmed, complete growth medium.
The number of cells were counted. Appropriate number of cells were transfer into a new centrifuge tube, pelleted by centrifugation at 90xg for 10 minutes, and resuspended in supplemented nucleofection solution.
About 200,000 cells in 15 ,1 nucleofection solution were mixed with about 7it1 of preformed SluCas9:sgRNA RNP complex.

[00289] Approximately 20 1 of the cell and RNP mix were transferred into a 16-well nucleofection strip tube, and then nucleofected with the DS-158 program in a 4D-Nucleofector (Lonza). Immediately after nucleofection, about 80 1 pre-warmed media were added into the each nucleocuvette and incubated at 37 degrees for 10 minutes. The contents (100)d) of each nucleocuvette were transferred into one well in a 12-well plate filled with 2m1 media and incubated at 37 degrees for 48 hours.

[00290] To determine cell viability 48 hours after nucleofection, the cells were stained with Hoechst and Propidium Iodide (Life Technologies). Cell viability was then assessed using ImageXpress Micro (Molecular Devices). In general, samples with an overall cell viability above 70%
were harvested and analyzed for indel analysis.

[00291] To isolate genomic DNA from HsMMs, the cells were washed with saline buffer, trypsinized and centrifuged. The cell pellets were treated with lysis buffer from the Maxwell RSC
Blood DNA Kit (Promega #AS1400), and genomic DNAs were extracted using a Maxwell RSC48 instrument (Promega #AS8500) according to the manufacturer's instruction. The concentrations of genomic DNAs were determined using QubitTM lx dsDNA HS Assay Kit (Thermo Fisher Scientific Q33231) according to the manufacturer's instruction.

[00292] To determine the gene editing efficiency, the genomic DNAs were amplified using primers flanking the DMD exon 45 genomic region. The following primer sequences were used:
MiSeq_hE45_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGgtattctgtettgtatcctttgg (SEQ
ID NO: 724) and MiSeq_hE45_R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGaatgttagtgccthcaccc (SEQ ID NO: 725).
The size of the amplicons was verified by analyze a small amount of the PCR
products on 2% E-gels (Thermo Fisher Scientific). A portion of the PCR product and the forward primer were then sent for sanger sequencing at Genewiz.

[00293] The sequencing results that pass the quality filter were used to determine editing efficiency and indel profile using the TIDE (Tracking of Indels by DEcomposition) algorithm. The following version of the algorithm was used for analysis https://shiny.vrtx.com/app/orrj/tide/ with the appropriate sgRNA sequence, the default analysis parameters, and a mock nucleofected sample as the control. Percentage of other insertions and deletions that have the potential to restore the reading frame of particular DMD patient mutations of interest were referred to as "RF
other". This represents the sum of 2, 5, 8, 11 bp deletions within the alignment window of -20bp to +20bp around the Cas9 cut site. The editing efficiency (% mutation) outputs for +lbp insertion, RF.
Other, and other indels from TIDE were then plotted using Prism 9.
2. Results

[00294] To optimize and improve gene editing efficiency of the top SluCas9 single-guide RNA
(sgRNA) candidates, different sgRNA scaffold sequences were tested in primary human skeletal muscle myoblasts (HsMM) using synthetic sgRNAs as shown in Table 11 below. Two spacer sequences were tested: E45SL23 (SEQ ID NO: 150 (DNA); SEQ ID NO: 930 (RNA)) and E455L24 (SEQ ID NO: 151 (DNA); SEQ ID NO: 931 (RNA)). Three scaffold sequences were tested: Slu-VCGT-4.5 (SEQ ID NO: 601 (DNA); SEQ ID NO: 918 (RNA)), Slu-VCGT-4 (SEQ ID NO:

(DNA); SEQ ID NO: 919 (RNA)), Slu-VCGT-5 (SEQ ID NO: 901 (DNA); SEQ ID NO: 920 (RNA)).
Table 11: Exemplary sgRNAs for testing sgRNA ID Scaffold Scaffold (RNA version) Spacer Spacer SEQ ID sgRNA
sequence (SEQ ID SEQ ID (RNA NO of NO of NO version) sgRNA
RNA) GGUAUCUUACAGGAA
VCGT-4.5 VCGT- GUGCUGGAAACAG UCUU CUCCAGGGUUUAAGU
4.5 (SEQ CACAGAAUCUACU ACAG ACUCUGUGCUGGAAA
ID NO: GAAACAAGACAAU GAAC CAGCACAGAAUCUAC
918) AUGUCGUGUUUAU UCCA
UGAAACAAGACAAUA
CCCAUCAAUUUAU GG UGUCGUGUUUAUCCC
UGGUGGGA
AUCAAUUUAUUGGUG
GGA

UUUUGGUAUCUUACA
VCGT-4.5 VCGT- GUGCUGGAAACAG GGUA GGAACUCGUUUAAGU
4.5 CACAGAAUCUACU UCUU
ACUCUGUGCUGGAAA
(SEQ ID GAAACAAGACAAU ACAG CAGCACAGAAUCUAC
NO: 919) AUGUCGUGUUUAU GAAC UGAAACAAGACAAUA
CCCAUCAAUUUAU UC UGUCGUGUUUAUCCC
UGGUGGGA
AUCAAUUUAUUGGUG
GGA
E455L23- Slu- GUUUCAGUACUCU 930 GGUA

(SEQ ID CACAGAAUCUACU ACAG ACUCUGUGCUGGAAA
NO: 920) GAAACAAGACAAU GAAC CAGCACAGAAUCUAC
AUGUCGUGUUUAU UCCA UGAAACAAGACAAUA
CCCAUCAAUUUAU GG UGUCGUGUUUAUCCC
UGGUGGGAU
AUCAAUUUAUUGGUG
GGAU

UUUUGGUAUCUUACA

(SEQ ID CACAGAAUCUACU UCUU ACUCUGUGCUGGAAA
NO: 921) GAAACAAGACAAU ACAG CAGCACAGAAUCUAC
AUGUCGUGUUUAU GAAC UGAAACAAGACAAUA

CCCAUCAAUUUAU UC
UGUCGUGUUUAUCCC
UGGUGGGAU
AUCAAUUUAUUGGUG
GGAU
E45SL23- Slu- GUUUcAGUACUCU 930 GGUA 928 GGUAUCUUACAGGAA

CUCCAGGGUUUcAGU
(SEQ ID ACUGAAACAAGAC ACAG
ACUCUGGAAACAGAA
NO: 922) AAUAUGUCGUGUU GAAC
UCUACUGAAACAAGA
UAUCCCAUCAAUU UCCA
CAAUAUGUCGUGUUU
UAUUGGUGGGAU GG
AUCCCAUCAAUUUAU
UGGUGGGAU
E455L24- Slu- GUUUcAGUACUCU 931 UUUU 929 UUUUGGUAUCUUACA

GGAACUCGUUUcAGU
(SEQ ID ACUGAAACAAGAC UCUU
ACUCUGGAAACAGAA
NO: 923) AAUAUGUCGUGUU ACAG
UCUACUGAAACAAGA
UAUCCCAUCAAUU GAAC
CAAUAUGUCGUGUUU
UAUUGGUGGGAU UC
AUCCCAUCAAUUUAU
UGGUGGGAU

[00295] The three scaffold sequences differ by the nucleotide identity, and thus the stem-loop Tin RNA secondary structure (FIG. 7A). In addition to differences in stem-loop I, Slu-VCGT-4.5 lacks the last nucleotide U at the 3' end of Stem 3 (not shown). The results indicate that, Slu-VCGT-5 scaffold produces higher editing efficiency compared to guides with a V4 or V4.5 scaffold in most conditions tested (shown in FIG. 7B).
Example 6 1. Materials and Methods

[00296] Transfection of HEK293FT cells

[00297] 293FT
cells were transfected in 12-well plates with 750 ng plasmid + 2.25 ILtL of Lipofectamine 2000. Three days after transfection, cells were trypsinized and sorted for GFP. GFP-positive cells were sorted directly into lysis buffer, and DNA extraction was performed using the Promega Maxwell RSC Blood DNA Kit. PCR was then performed on the DNA using exon-specific primers that targeted the relevant cut site.

[00298] Transfection of N2a cells

[00299] N2a (Neuro2a) cells were transfected in 12-well plates after 24-hour of growth with 1000ng plasmid + 3 ILtL of Lipofectamine 2000. Three days after transfection, cell were trypsinized and sorted for GFP (green fluroescent protein). GFP-positive cells were sorted via FACS
(flurorescence-activated cell sorting) directly into lysis buffer, and DNA
extraction was performed using the Promega Maxwell RSC Blood DNA Kit. PCR was then performed on the DNA
using exon-specific primers that targeted the relevant cut site.

[00300] Amplicon deep sequencing library preparation for HEK293FT cells

[00301] Genomic DNA were extracted after sorting for 100k GFP positive cells and were subjected for amplification by locus specific amplicon. The amplified products were purified by AMPure beads (0.8x) followed by QC with 1% E-gel and concentrations were measured using QuBiT
for normalization of samples. Barcoding PCR was carried out with i5 and i7 indices and they were purified by 0.7x AMPure beads followed by QC with Tapestation or 1% E-gel. The barcoded samples were then measured by QuBiT and based on the concentration, the samples were pooled and subjected for library preparation for loading into MiSeq. 8pM of library was loaded with 20% PhiX spike-in and the output were transferred to the Computational Genomics team for assessing the indel efficiencies for the guides.

[00302] Human primary skeletal muscle myoblasts (HsMMs) culture

[00303] On day zero, two frozen vials of HsMMs (lot 20TL070666) was thawed and grown in complete growth media in a 37 C, 5% CO2, humidified incubator in two T75 flasks. The following day, on day 1, the media was changed. On day 3, the cells were passaged such that there were 9x10 cells seeded into 7 T175 flasks. On day 4, the media was changed in each flask. On day 6, RNP
nucleofection was performed.

[00304] Nucleofection of HsMM cells

[00305] HsMM cells were nucleofected with RNP using the Lonza 4D
nucleofector. For each sample, 7 jaL of RNP were combined with 0.3e6 cells in 15 jaL P5 solution. RNP
was prepared in a 6:1 gRNA:Cas9 ratio at various concentrations. For dual-cut samples that contain 2 gRNAs, RNP was pre-formed with a single gRNA first. RNP was formed by incubating gRNA and protein for 20 min at room temperature. After electroporation, 80 jaL of complete growth media was added to each sample and samples were incubated in a 37 C, 5% CO2, humidified incubator. After 10 minutes, the samples were transferred to 12-well plates containing 2 mL of complete growth media that had been previously equilibrated in a 37 C, 5% CO2, humidified incubator.

[00306] Cell harvesting and gDNA extraction

[00307] To determine cell viability 48 hours after nucleofection, the cells were stained with Hoechst and Propidium Iodide (Life Technologies). Cell viability was then assessed using ImageXpress Micro (Molecular Devices). In general, samples with an overall cell viability above 70%
were harvested and analyzed for indel analysis. To isolate genomic DNA from HsMMs, the cells were washed with saline buffer, trypsinized and centrifuged. The cell pellets were treated with lysis buffer from the Maxwell RSC Blood DNA Kit (Promega #AS1400), and genomic DNAs were extracted using a Maxwell RSC48 instrument (Promega #AS8500) according to the manufacturer's instruction. The concentrations of genomic DNAs were determined using QubitTM
lx dsDNA HS
Assay Kit (Thermo Fisher Scientific Q33231) according to the manufacturer's instruction.

[00308] Amplicon deep sequencing library preparation for HsMM

[00309] To determine the gene editing efficiency, the genomic DNAs were amplified using primers flanking the DMD exon 45 genomic region. The following primer sequences were used:
MiSeq_hE45_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGgtcffictgtcttgtatcctttgg (SEQ
ID NO: 724) and MiSeq_hE45_R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGaatgttagtgccthcaccc (SEQ ID NO: 725).
The size of the amplicons was verified by analyze a small amount of the PCR
products on 2% E-gels (Thermo Fisher Scientific). The PCR product was purified by AMPure XP beads (A63881). The purified PCR product was amplified again with primers that contain barcodes and Illumina adaptors.
Multiple barcoded samples were pooled, combined with PhiX library, and loaded onto the Illumina Mi-Seq platform. MiSeq Reagent Kit v3 (MS-102-3003) was used to produce a 600-cycle run.

[00310] Maintenance and differentiation of C2C12 myotubes

[00311] C2C12 are maintained in DMEM supplemented with 10% FBS (Fetal Bovine Serum) and 1% Pen/Strep. For culture purposes, cells should not be allowed to reach >75% confluency.
C2C12 myoblasts are differentiated in DMEM supplemented with 2% HS and 1%
Pen/Strep. Briefly, cells are seeded at 42-45k/cm2 and differentiation is started 24hr after seeding (once cells reach ¨90-95% confluency). Differentiation medium is changed on day 1 and then refreshed every other day.

[00312] Transduction of C2C12 myotubes

[00313] C2C12 myoblasts were allowed to differentiate to myotubes in differentiation medium (DMEM with 2% horse serum and 1% Pen/Strep) for 6 days. Two hours before viral transduction, myotubes were treated with Neuraminidase type III (Sigma-Aldrich, 50 mU/m1), followed by washing with differentiation medium twice. Myotubes were incubated with AAV (MOI
1.0E7) and centrifuged at 1000xg at 4C for 1.5 hours. After spin transduction, the virus is aspirated, and the myotubes are washed with cold PBS once followed by two washes with differentiation medium.
The myotubes are cultured in differentiation medium for an additional week (7 days) before being harvested for INDEL
analysis (ICE/NGS) or fixed for immunohistochemistry analysis (IHC). DNA/RNA
extraction was performed using the Qiagen AllPrep DNA/RNA Minikit.

[00314] Cas9 nuclear localization by immunofluorescence staining of C2C12 myotubes

[00315] 7 days post AAV transduction (day 12 of myotube differentiation) C2C12 myotubes were fixed with 4% paraformaldehyde (PFA) for 20 mins at room temperature (RT).
After fixation, cells were washed twice with PBS followed by permeabilization with PBS-0.5% Triton for 15 mins at RT.
Prior to incubation with primary antibodies cells were incubated with a blocking buffer for 30 mins at RT (PBS+10%FBS+0.1%Triton). Cell were then incubated with primary antibodies in block for 2 hours at RT (or overnight at 4 C) followed by 3 washes with PBS-Tween 0.1%.
Secondary antibodies are added for 1.5-2 hours at RT. Antibodies for these studies are listed here at Table 12:

Table 12:
Primary Antibodies Host Dilution SluCas9 (GenScript 11E3 A9-1) Mouse 1:100 SaCas9 (Diagenode C15200230-100) Mouse 1:400 My oG Rabbit 1:200 Secondary Antibodies Host Dilution Anti-mouse 488 1:500 Anti-Rabbit 594 1:500

[00316] Vector genome quantitation

[00317] Quantitative polymerase chain reaction (qPCR) was used to quantify levels of AAV9 DNA in C2C12 differentiated cells. gDNA was extracted using the Qiagen AllPrep kit and quantified using the Qubit 4 Fluorometer and diluted to a final concentration of 2.5 ng/u.L.

[00318] Absolute quantification of AAV9 vectors was performed by constructing a standard curve prepared from known quality of linearized plasmids encoding the region of interest. A set of Quality control (QC) were included to validate the methods of quantification. A set of non-template control (NTC) samples were included to confirm the specificity of reactions.

[00319] qPCR reactions were conducted in triplicate using a QuantStudio 6 Flex Real-Time PCR
System (Thermo Fisher). A linear regression analysis was performed using threshold cycle (Ct) values of the standard curve. From this linear regression, the Ct values of the samples were used to quantify the number of copies per jug of gDNA of AAV9 present in each sample.

[00320] Cas9 and sgRNA transgene expression

[00321] Reverse transcription polymerase chain reaction (RT-qPCR) was used to quantify levels of Cas9 mRNA and the expression of gRNA in C2C12 differentiated cells. RNA was extracted using the Qiagen AllPrep kit and extracted RNA were quantified using the Qubit 4 Fluorometer and diluted to a final concentration of 20 ng/u.L. Absolute quantification of gRNA and Cas9 mRNA were performed by constructing a standard curve prepared from known quality of a T7 transcript encoding the corresponding region of interest. A set of Quality control (QC) were included to validate the methods of quantification. A set of non-template control (NTC) samples were included to confirm the specificity of reactions.

[00322] RT-qPCR reactions were conducted in triplicate using a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher). A linear regression analysis was performed using threshold cycle (Ct) values of the standard curve. From this linear regression, the Ct values of the samples were used to quantify the number of copies per jig of RNA of either Cas9 mRNA or gRNA
present in samples.

[00323] Amplicon deep sequencing library preparation for C2C12

[00324] gDNA was extracted using the Qiagen AllPrep kit. The relevant locus for exon 45 of mouse Dmd was amplified by PCR and amplified products were purified by AMPure beads (0.8x) followed by QC with 1% E-gel. DNA concentrations were measured using QuBiT for normalization of samples and Illumina sequencing libraries were created from these PCR
products using a MiSeq Reagent Kit v3 (600-cycle).

[00325] Next generation sequencing data analysis

[00326] A custom bioinformatic workflow was used to process the Illumina sequencing data.
First, poor quality reads were removed. Reads that passed the quality filter were trimmed using Trimmomatic to remove adapters and low quality bases. Then reads mapping to the PhiX genome were removed, and paired end reads were merged using PEAR. Based on the expected cut sites corresponding to the two guides, three reference amplicon sequences were created ¨wildtype amplicon, deletion amplicon with a deletion between the cutsites for the two guides, and inversion amplicon with an inversion of the sequence between the two the cutsites for the two guides. The merged reads were assigned to one of three amplicons (wild-type, deletion, inversion) based on the alignment score provided by the Needleman Wunsch algorithm as implemented in ParasailNeedle.
The CIGAR string provided by the alignment algorithm was parsed to identify the sequence of indel events. The summarized table of indel events and their frequencies was overlaid with information on exon length, exon frame, and position of the premature stop codon introduced as a result of the DMD
disease causing mutation, to characterize Cas9-associated indel events as productive (e.g., precise deletion, RF +1, RF Other, Exon skipping) or non-productive (e.g., OE) (Table 13). Throughout this workflow, a stringent set of QQC criteria was applied to filter poor quality samples. (Table 13).
Table 13:
Indel group Ranking Definition Order Wild-type amplicon NE 1 insertion = 0 & deletion = 0 RF +1 2 1-nt insertion at a reframe guide 3n+1 deletion within the exon boundary at the only reframe guide no edits at the exon guide RF Other 3 aggregated 3n+1 deletion within the exon boundary at the two reframe guides < 17-nt insertion at the only reframe guide no edits at the exon gate < 17-nt aggregated 3n+1 insertion at the two reframe guides indels overlap with the splice acceptor (AG) (5'-end of the target exon) E deletion with > 9-nt overlap with the splicing window at one of the two guides xon skipping 4 insertion at the exact GT/AG splicing sites and with length > 9-nt at one of the two guides OE 5 Other indels Deletion amplicon Precise 1 segmental deletion between the two cut sites deletion (Rest) 2 SingleCut indel profiling with 3n phase Inversion amplicon OE 1 all indels

[00327] Animal and study design

[00328] This study was designed to evaluate all-in-one gene editing vector candidates that have been created to include both the sgRNA and a Cas9 endonuclease to mediate gene editing at the DMD
locus. In this study, the efficacy of the one-vector gene editing candidates was assessed in vivo by measuring dystrophin restoration, on-target gene editing, tissue vector genomes, and Cas9 and sgRNA
transgene expression following an intraperitoneal administration to dEx44 mice at postnatal day 4 or 5.

[00329] Table 14A describes the in-vivo study design:
;
Group Mouse . Test Article Vector # of Dose Sample Collection and # Strain ; Age Test Article 1 Configuration 1 Animals (vg/kg) Analysis ;

; ............... Vehicle N/A
2 ; 6 N/A
1 3 EX 145 1 AAV9-Cas9 : AAV9-M-sgRNA- ;
145 ; 8 1 x 10'4 : 1 x 10'4 ; 4 AAV9-E45S124 Slu24:2XNLS:7SK-Hlm 8 2 x iota Tissue collection at 4 ................................................ õ ..... weeks post-dose AAV9-vVT046 Slu18/4:2XNLS+:hU6-hU6 8 2 x 10'4 Collect: heart, TA, ; 6 AAV9-vVT047 ; Slu18/4:3XNLS:hU6-hU6 ; 8 2 x 10'4 quadriceps, triceps, diaphragm, and liver PND4 to ; AAV9-vVT048 ; Slu18/4:3XNLS:hU6-7SK ; 8 2 x 10' AEx44 PND5 ......................................... 4 Analyses: Dystrophin 7 ................................................ õ ..
; 8 AAV9-vVT052 ; Slu21/7:2XNLS+:7SK-H1m ; 8 2 x 1014 restoration, on-target gene editing, Cas9 protein ; 9 AAV9-vVT054 ; Slu24:3XNLS:7SK-H1m ; 8 2 x 10'4 expression, vector genome copy number, and transgene expression on AAV9-vVT049 ; Slu18/4:3XNLS:H1m-M11 ; 8 ; 2 x 10'4 select tissues 1' t 1¨

; 11 AAV9-vVT050B ; Slu18/4:3XNLS:7SK-Him ; 8 , 2 x 10'4 13 AAV9-vVT051 Slu18/4:2XNLS+:7SK- Hlm 8 2 x 10'4 ' SaKKH10/20:2XNLS+:7SK- ;
14 AAV9-vVT053B ; 8 2 x 10'4 : Him , WT = wildtype PND4 to PND5 = postnatal day 4 to postnatal day 5 N/A = not applicable

[00330] Amplicon deep sequencing library preparation for in vivo study

[00331] gDNA was extracted from mouse heart, quadricep, and triceps tissue using the Maxwell RSC Tissue DNA Kit and quantified via Qubit. The relevant locus of exon 45 of the mouse DMD
gene was amplified using PCR. PCR products were visualized using both E-gel and TapeStation to confirm proper size and were purified using AMPure XP beads. PCR products then underwent a second PCR reaction to add unique 5' and 3' barcodes corresponding to each sample. These PCR
products were quantified via Qubit and normalized to 4 nM each. The normalized products were then pooled, combined with a PhiX library to increase diversity, and loaded onto a MiSeq instrument. The library was sequenced using a MiSeq Reagent Kit v3 (600-cycle) and raw data was transferred to the DCS team.

[00332] Next generation sequencing data analysis for in vivo study

[00333] A custom bioinformatic workflow was used to process the Illumina sequencing data.
First, poor quality reads (below Q30) were removed. The surviving reads were trimmed using using Trimmomatic Trimmomatic to remove adapters and low quality bases. Then reads mapping to the PhiX genome were removed, and paired end reads were merged using PEAR. Based on the expected cut sites corresponding to the two guides, three reference amplicon sequences were created ¨
wildtype amplicon, deletion amplicon with a deletion between the cut sites for the two guides, and inversion amplicon with an inversion of the sequence between the two the cut sites for the two guides.
The merged reads were assigned to one of three amplicons (wild-type, deletion, inversion) based on the alignment score provided by the Needleman Wunsch algorithm as implemented in ParasailNeedle.
The CIGAR string provided by the alignment algorithm was parsed to identify the sequence of indel events. The summarized table of indel events and their frequencies was overlaid with information on exon length, exon frame, and position of the premature stop codon introduced as a result of the DMD
disease causing mutation, to characterize Cas9-associated indel events as productive (e.g., precise deletion, RF +1, RF Other, Exon skipping) or non-productive (e.g., OE) (Table 1). Throughout this workflow, a stringent set of QC criteria was applied to filter poor quality samples and all samples are checked for potential contaminations from other treatment groups in the same study.
2. Results

[00334] Dual cut sgRNA in vitro screening

[00335] The average indel frequency of sgRNAs targeting exon 45 was tested in primary human skeletal muscle myoblasts (HsMM) (Figure 8), with a high-performing SpCas9 sgRNA (E45Sp52) included as a reference. Each of the five SluCas9 sgRNA pairs evaluated showed an average precise segmental deletion frequency higher than 60% (Figure 8). The sequences of the spacers for the guide RNAs used in Figure 8 (i.e., 18, 4, and 18+4; 21, 7, and 21+7; 17, 22, and 17+22; 17, 23 and 17+23;
and 19, 21, and 19+21) are shown in Table 10.

[00336] The average indel frequency of sgRNAs targeting exon 51 was determined in HEK293FT
cells (Figure 9), which shows an indel frequency higher than 50% and an average precise segmental deletion frequency above 50% for E51SaKKH20/9, E51SaKKH20/27, E51SL10/3, E51SL31/5, E51SL31/7, E51SL31/8, E51SL10/16, E51SL23/31 and E51SL10/24 in HEK293FT cells.
Table 14B
shows the sgRNA ID and the spacer sequence for each sgRNA pairing shown in Figure 9, where the pairings shown on the X-axis in Figure 9 (e.g., 2 + 6) are within the sgRNA ID
as the last character of each term (e.g., E5 1 Sa2 E5 1 Sa6).

Table 14B:
sgRNA ID Protospacer sequence (a comma separates the two spacers) xroaroweroa4 E51Sa2 E51Sa6 GTTGTGTCACCAGAGTAACAG (SEQ ID NO: 20), TTGATCAAGTTATAAAATCACA (SEQ ID NO: 24) E51SaCas9KKH4 E51SaCas9KKH43 ATGATCATCTCGTTGATATCCT (SEQ ID NOS: 1022, 2031), TAGCTCCTACTCAGACTGTTAC (SEQ ID NO: 1055) E51SaCas9KKH20 E51SaCas9KKH5 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), ......................... AAGGTCACCCACCATCACCCTC (SEQ ID NO: 1023) E51SaCas9KKH43 E51SaCas9KKH6 TAGCTCCTACTCAGACTGTTAC (SEQ ID NO: 1055), ATCACCCTCTGTGATTTTATAA (SEQ ID NO: 1024) E51SaCas9KKH43 E51SaCas9KKH7 TAGCTCCTACTCAGACTGTTAC (SEQ ID NO: 1055), ......................... ATAACTTGATCAAGCAGAGAAA (SEQ ID NO: 1025) E51SaCas9KKH20 E51SaCas9KKH9 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), ATCAAGCAGAGAAAGCCAGTCG (SEQ ID NO: 1027) E5 1 SaCas9KKH10 E51SaCas9KKH43 CAGTCGGTAAGTTCTGTCCAAG (SEQ ID NO: 1028), ......................... TAGCTCCTACTCAGACTGTTAC (SEQ ID NO: 1055) E51SaCas9KKH11 E51SaCas9KKH43 GTAAGTTCTGTCCAAGCCCGGT (SEQ ID NO: 1029), TAGCTCCTACTCAGACTGTTAC (SEQ ID NO: 1055) E51SaCas9KKH12 E51SaCas9KKH20 AGCCCGGTTGAAATCTGCCAGA (SEQ ID NOS: 1030, 2041), ......................... CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037) E51SaCas9KKH13 E51SaCas9KKH20 AGCAGGTACCTCCAACATCAAG (SEQ ID NOS: 1031, 2043), CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037) E5 1 SaCas9KKH14 E51SaCas9KKH20 CAACATCAAGGAAGATGGCATT (SEQ ID NO: 1032), CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037) E51SaCas9KKH20 E51SaCas9KKH27 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), TCAACGAGATGATCATCAAGCA (SEQ ID NOS: 1042, 2060) E51SaCas9KKH20 E51SaCas9KKH28 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), GTGACCTTGAGGATATCAACGA (SEQ ID NO: 1043) E51SaCas9KKH20 E51SaCas9KKH29 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), TGGGTGACCTTGAGGATATCAA (SEQ ID NOS: 1044, 2063) E51SaCas9KKH20 E51SaCas9KKH32 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), AAGTTATAAAATCACAGAGGGT (SEQ ID NO: 1045) E51SaCas9KKH20 E51SaCas9KKH33 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), ATCAAGTTATAAAATCACAGAG (SEQ ID NO: 1046) E51SaCas9KKH20 E51SaCas9KKH34 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), TTGATCAAGTTATAAAATCACA (SEQ ID NO: 24) E5 1 SES E51SaCas9KKH43 CTTTCTCTGCTTGATCAAGTTA (SEQ ID NO: 1047), TAGCTCCTACTCAGACTGTTAC (SEQ ID NO: 1055) E51SaCas9KKH20 E51SaCas9KKH36 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), CCGACTGGCTTTCTCTGCTTGA (SEQ ID NO: 1048) E51SaCas9KKH20 E51SaCas9KKH39 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), AAATGCCATCTTCCTTGATGTT (SEQ ID NOS: 1051, 2070) E51SaCas9KKH20 E51SaCas9KKH41 CCACAGGTTGTGTCACCAGAGT (SEQ ID NO: 1037), AGGAAACTGCCATCTCCAAACT (SEQ ID NO: 1053) E51SL1 E51SL10 TGATCATCTCGTTGATATCCTC (SEQ ID NO: 170), GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179) E51SL2 E51SL31 TTGATCAAGCAGAGAAAGCCAG (SEQ ID NO: 171), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E51SL10 E51 SL3 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), ------------------------- AGTCGGTAAGTTCTGTCCAAGC (SEQ ID NO: 172) E51SL31 E51SL5 AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200), ------------------------- CAGAGCAGGTACCTCCAACATC (SEQ ID NO: 174) E51SL31 E51SL7 AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200), ------------------------- CAAGGAAGATGGCATTTCTAGT (SEQ ID NO: 176) E51SL31 E51SL8 AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200), AGATGGCATTTCTAGTTTGGAG (SEQ ID NO: 177) E51SL10 E51SL15 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), CAACGAGATGATCATCAAGCAG (SEQ ID NO: 184) E51SL10 E51SL16 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), GAGGGTGATGGTGGGTGACCTT (SEQ ID NOS: 22, 185) E51SL10 E51SL18 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), TATAAAATCACAGAGGGTGATG (SEQ ID NOS: 23, 187) E51SL10 E51SL19 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), AGTTATAAAATCACAGAGGGTG (SEQ ID NO: 188) E51SL10 E51 SL20 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), TGATCAAGTTATAAAATCACAG (SEQ ID NO: 189) E515L21 E515L31 TTGATCAAGTTATAAAATCACA (SEQ ID NO: 24), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E51 SL22 E51 SL31 GGGCTTGGACAGAACTTACCGA (SEQ ID NO: 191), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E51 SL23 E51 SL31 CTCTGGCAGATTTCAACCGGGC (SEQ ID NO: 192), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E515L10 E51 SL24 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), ACCTGCTCTGGCAGATTTCAAC (SEQ ID NO: 193) E51 SL25 E51 SL31 TACCTGCTCTGGCAGATTTCAA (SEQ ID NO: 194), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E51SL 10 E51SL26 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), CTTGATGTTGGAGGTACCTGCT (SEQ ID NO: 195) E51SL10 E515L27 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), ------------------------- AATGCCATCTTCCTTGATGTTG (SEQID NO: 196) E51SL10 E51 SL28 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179), ------------------------- AGAAATGCCATCTTCCTTGATG (SEQ ID NO: 197) E51SLCas9KH26 E51SLCas9KH73 GATGGCAGTTTCCTTAGTAACC (SEQ ID NOS: 1036, 2048), ------------------------- AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E5 1 SLCas9KH25 E5 1 SLCas9KH73 TAGTTTGGAGATGGCAGTTTCC (SEQ ID NO: 2047), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E5 1 SLCas9KH24 E5 1 SLCas9KH73 TGGCATTTCTAGTTTGGAGATG (SEQ ID NO: 2046), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E5 1 SLCas9KH21 E51SLCas9KH73 CAAGGAAGATGGCATTTCTAGT (SEQ ID NO: 176), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E5 1 SLCas9KH20 E51SLCas9KH30 AACATCAAGGAAGATGGCATTT (SEQ ID NO: 2044), CACAGGTTGTGTCACCAGAGTA (SEQ ID NO: 2051) E5 1 SLCas9KH19 E51SLCas9KH30 GGTACCTCCAACATCAAGGAAG (SEQ ID NO: 175), CACAGGTTGTGTCACCAGAGTA (SEQ ID NO: 2051) E5 1 SLCas9KH20 E51 SL Cas9KH32 AACATCAAGGAAGATGGCATTT (SEQ ID NO: 2044), TGTCACCAGAGTAACAGTCTGA (SEQ ID NO: 2053) E5 1 SLCas9KH20 E51 SL Cas9KH34 AACATCAAGGAAGATGGCATTT (SEQ ID NO: 2044), CACCAGAGTAACAGTCTGAGTA (SEQ ID NO: 2054) E51SLCas9KH18 E51SLCas9KH73 AGCAGGTACCTCCAACATCAAG (SEQ ID NOS: 1031, 2043), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E5 1 SLCas9KH17_E51SLCas9KH73 CAGAGCAGGTACCTCCAACATC (SEQ ID NO: 174), AGCTCCTACTCAGACTGTTACT (SEQ ID NO: 200) E51Sa2 GTTGTGTCACCAGAGTAACAGT (SEQ ID NO: 20) E515L10 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179) E51S1uCas9-2 TTGATCAAGCAGAGAAAGCCAG (SEQ ID NO: 171) E51S1uCas9-10 GTCACCAGAGTAACAGTCTGAG (SEQ ID NO: 179)

[00337] AAV configurations for the dual cut single vector candidates

[00338] A combination of promoter orientations, and configurations, NLS
sequences and sgRNA
scaffolds were selected for generating AAV plasmids and evaluation on sgRNA
transgene expression, AAV manufacturability, and editing efficiency in vitro and in vivo. AAV
plasmid configurations are listed in Table 15 and Table 16. Promoter, NLS and scaffold sequences are listed in Table 17.

[00339] Guide RNA were prepared according to standard methods in a single guide (sgRNA) format. A single AAV vector was prepared that expresses the guide RNA pair and a variant SaCas9 or the guide RNA pair and a variant SluCas9. The AAV vectors were administered to C2C12 cells in vitro and to mice (e.g., dEx44 mice) in vivo to assess the ability of the AAV
to express the guide RNA and Cas9, edit the targeted exon, and reframe the dystrophin gene (in vivo studies only).
Table 15:
Pol III Orientation Configuration NLS1 Endonuclease NLS2 NLS3 Scaffold Promoter = 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-GSGS Nucleoplasmin- V5 hU6c GSVD GSGS
= c> 4 hU6c-Cas9- c-Myc- SaCas9-KKH
SV-40-GSGS Nucleoplasmin- V2 hU6c GSVD GSGS
= 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-MCS- Nucleoplasmin- V5 hU6c GSVD GSGS GSGS
=> 4 hU6c-Cas9- c-Myc- SaCas9-KKH SV-40-MCS-Nucleoplasmin- V2 hU6c GSVD GSGS GSGS
= 4 hU6c-Cas9- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 hU6c = 4 hU6c-Cas9- SV-40 (+) SaCas9-KKH Nucleoplasmin N/A V2 hU6c ,<¨ hU6c-hU6c- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 Cas9 GSVD GSGS
, hU6c-hU6c- c-Myc- SaCas9-KKH SV-40-GSGS Nucleoplasmin- V2 Cas9 GSVD GSGS
,<¨ hU6c-hU6c- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 hU6c:hU6c GSGS
Cas9 GSVD GSGS
hU6c-hU6c- c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V2 Cas9 GSVD GSGS GSGS
hU6c-hU6c- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 Cas9 hU6c-hU6c- SV-40 (+) SaCas9-KKH
Nucleoplasmin N/A V2 Cas9 4 4 hU6c-hU6c- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 Cas9 GSVD GSGS
4 4 hU6c-hU6c- c-Myc- SaCas9-KKH SV-40-GSGS
Nucleoplasmin- V2 Cas9 GSVD GSGS
4 4 hU6c-hU6c- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 Cas9 GSVD GSGS GSGS
4 4 hU6c-hU6c- c-Myc- SaCas9-KKH SV-40-MCS-Nucleoplasmin- V2 Cas9 GSVD GSGS GSGS
4 4 hU6c-hU6c- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 Cas9 4 4 => hU6c-hU6c- SV-40 (+) SaCas9-KKH
Nucleoplasmin N/A V2 Cas9 , => 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 <¨ c> 4 hU6c-Cas9- c-Myc- SaCas9-KKH SV-40-GSGS
Nucleoplasmin- V2 <¨ => 4 hU6c-Cas9- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 , => 4 hU6c-Cas9- c-Myc- SaCas9-KKH SV-40-MCS-Nucleoplasmin- V2 hU6c-7SK2- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 Cas9 GSVD GSGS GSGS
hU6c-7SK2- c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V2 Cas9 GSVD GSGS GSGS
hU6c-7SK2- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 Cas9 GSVD GSGS
, => hU6c-7SK2- c-Myc- SaCas9-KKH SV-40-GSGS
Nucleoplasmin- V2 Cas9 GSVD GSGS
hU6c:7SK2 hU6c-7SK2- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 Cas9 hU6c-7SK2- SV-40 (+) SaCas9-KKH Nucleoplasmin N/A V2 Cas9 4 4 => hU6c-7SK2- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 Cas9 GSVD GSGS GSGS
4 4 => hU6c-7SK2- c-Myc- SaCas9-KKH SV-40-MCS-Nucleoplasmin- V2 Cas9 GSVD GSGS GSGS
4 4 => hU6c-7SK2- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 Cas9 GSVD GSGS
4 4 c> hU6c-7SK2- c-Myc- SaCas9-KKH SV-40-GSGS
Nucleoplasmin- V2 Cas9 GSVD GSGS
4 4 => hU6c-7SK2- SV-40 (+) S1uCas9 Nucleoplasmin Cas9 4 4 => hU6c-7SK2- SV-40 (+) SaCas9-KKH
Nucleoplasmin N/A V2 Cas9 <¨ => 4 7SK2-Cas9- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 hU6c GSVD GSGS
, => 4 7SK2-Cas9- c-Myc- SaCas9-KKH SV-40-GSGS
Nucleoplasmin- V2 hU6c GSVD GSGS
7SK2:hU6c <¨ => 4 7SK2-Cas9- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 hU6c GSVD GSGS GSGS
<¨ c> 4 7SK2-Cas9- c-Myc- SaCas9-KKH SV-40-MCS-Nucleoplasmin- V2 hU6c GSVD GSGS GSGS
7SK2-hU6c- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 Cas9 GSVD GSGS GSGS
7SK2-hU6c- c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V2 Cas9 GSVD GSGS GSGS
7SK2-hU6c- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 Cas9 GSVD GSGS
, => 7SK2-hU6c- c-Myc- SaCas9-KKH SV-40-GSGS
Nucleoplasmin- V2 Cas9 GSVD GSGS
7SK2-hU6c- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 Cas9 7SK2-hU6c- SV-40 (+) SaCas9-KKH Nucleoplasmin N/A V2 Cas9 4 4 => 7SK2-hU6c- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 Cas9 GSVD GSGS GSGS
4 4 => 7SK2-hU6c- c-Myc- SaCas9-KKH SV-40-MCS-Nucleoplasmin- V2 Cas9 GSVD GSGS GSGS
4 4 => 7SK2-hU6c- c-Myc- S1uCas9 SV-40-GSGS
Nucleoplasmin- V5 Cas9 GSVD GSGS
4 4 => 7SK2-hU6c- c-Myc- SaCas9-KKH SV-40-GSGS
Nucleoplasmin- V2 Cas9 GSVD GSGS
4 4 => 7SK2-hU6c- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 Cas9 4 4 => 7SK2-hU6c- SV-40 (+) SaCas9-KKH Nucleoplasmin N/A V2 Cas9 , => 4 hU6c-Cas9-Him c-Myc- S1uCas9 SV-40-GSGS Nucleoplasmin-GSVD GSGS
hU6c:Hlm , => 4 hU6c-Cas9-Him c-Myc- SaCas9-KKH SV-40-GSGS Nucleoplasmin-GSVD GSGS
<¨ => 4 7SK2-Cas9- SV-40-GS S1uCas9 Nucleoplasmin N/A V5 Him <¨ => 4 7SK2-Cas9- SV-40-GS SaCas9-KKH Nucleoplasmin N/A V2 Him <¨ => 4 7SK2-Cas9- SV-40 (+) S1uCas9 Nucleoplasmin N/A V5 Him <¨ => 4 7SK2-Cas9- SV-40 (+) SaCas9-KKH
Nucleoplasmin N/A V2 7SK2:Hlm Him <¨ => 4 7SK2-Cas9- c-Myc- S1uCas9 SV-40-MCS-Nucleoplasmin- V5 Him GSVD GSGS GSGS
<¨ => 4 7SK2-Cas9- c-Myc- SaCas9-KKH SV-40-MCS-Nucleoplasmin- V2 Him GSVD GSGS GSGS
<¨ => 4 7SK2-Cas9- c-Myc- SaCas9 SV-40-MCS-Nucleoplasmin- V2 Him GSVD GSGS GSGS

=> H1m-Cas9-M11 c-Myc- SluCas9 SV-40-MCS- Nucleoplasmin- V5 GSVD GSGS GSGS
Hlm:M11 H1m-Cas9-M11 c-Myc- SaCas9-KKH SV-40-MCS- Nucleoplasmin- V2 GSVD GSGS GSGS
Table 16:
Pol In Promoter Orientation Configuration hU6:1-11:7SK --- hU6-H1-7SK1 hU6:7SK 4 0 4 hU6-Stuffer-7SK1 hU6:7SK 4 4 0 hU6-7SK-Stuffer hU6c:hU6c <¨ 0 4 hU6c-Stuffer-hU6c Self-complementary AAV hU6c:mU6 <¨ 0 4 hU6c-Stuffer-mU6 7SK2:hU6c:hU6c:7SK2 0 4 7SK2-hU6c-Stuffer-hU6c-7SK2 7SK2:hU6c:mU6:7SK2 <¨ 0 4 7SK2-hU6c-Stuffer-mU6c-7SK2 7SK2:hU6c:hU6c:7SK2 0 4 7SK2-hU6c-Stuffer-hU6c-7SK2 7SK2:hU6c:mU6:7SK2 <¨ 0 4 7SK2-hU6c-Stuffer-mU6-7SK2 ...............................................................................
...............................................................................
..........................................
...............................................................................
...............................................................................
..........................................
hU6c:hU6c <¨ 0 4 hU6c-Stuffer-hU6c hU6c:mU6 <¨ 0 4 hU6c-Stuffer-mU6 7SK2:hU6c:hU6c:7SK2 0 4 7SK2-hU6c-Stuffer-hU6c-7SK2 Single-stranded AAV
75K2:hU6c:mU6:75K2 <¨ 0 4 75K2-hU6c-Stuffer-mU6c-75K2 75K2:hU6c:hU6c:75K2 0 4 75K2-hU6c-Stuffer-hU6c-75K2 75K2:hU6c:mU6:75K2 <¨ 0 4 75K2-hU6c-Stuffer-mU6-75K2 Table 17:
Promoter Sequences hU6c GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGAT
AATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGT
AATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTT
ACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACAC
C (SEQ ID NO: 705) CAAAACAGCCGG
AAAT CAAGT CCGT TTAT CT CAAACT T TAGCAT TT T GGGAATAAAT GATAT T T GCTAT GCT GG

T TAAAT TAGAT TT TAGT TAAATT T CCT GCT GAAGCT CTAGTAC GATAAGCAACTT GACCTAA
GT GTAAAGT T GAGACTT CCTT CAGGT TTATATAGCT T GT GCGCCGCTT GGGTACCT C ( SEQ
ID NO: 706) Him AATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTT
GGGAATCTTATAAGTTCTGTATGAGACCACTCTTTCCC (SEQ ID NO: 707) TCAGGTTTATATAGTTCTGTATGAGACCACTCTTTCCC (SEQ ID NO: 708) hU6 ccCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTT
CGCGTCGCGCAGACACT GCTCGGTAGTTTCGGGGATCAGCGTTT GAGTAAGAGCCCGCGTCT
GAACCCTCCGCGCCGCCCCGGCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACG
GAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGA
TTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACT
GTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTT
TGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATT
TCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC (SEQ ID NO: 42) mU6 Gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgggagaagct cggctactcccctgccccggttaatttgcatataatatttcctagtaactatagaggcttaa tgtgcgataaaagacagataatctgttctttttaatactagctacattttacatgataggct tggatttctataagagatacaaatactaaattattattttaaaaaacagcacaaaaggaaac tcaccctaactgtaaagtaattgtgtgttttgagactataaatatcccttggagaaaagcct tgtt (SEQ ID NO: 43) GGGAATCTTATAAGTTCTGTATGAGACCACGGTACACC (SEQ ID NO: 44) AAATCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGG
TTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAA
GTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTAC (SEQ ID
NO: 45) NLS-linker AA Sequences c-Myc-GSVD .. PLAKKKKLDGSVD (SEQ ID NO: 36) SV-40-GS PKKKRKVGS (SEQ ID NO: 37) SV-40 (+) PKKKRKVGIHGVPAA (SEQ ID NO: 38) SV-40-GSGS GSGSPKKKRKV (SEQ ID NO: 39) TGGGPGGGAAAGSGSPKKKRKV (SEQ ID NO: 40) GSGS
Nucleoplasmin-GSGSKRPAATKKAGQAKKKK (SEQ ID NO: 41) GSGS
Scaffold Sequences SluCas9 scaffold GUUUCAGUACU CU GGAAACAGAAUCUACUGAAACAAGACAAUAU GU C GUGUUUAU
C C CAUCA
V5 AUUUAUUGGUGGGAU (SEQ ID NO: 922) SaCas9 scaffold GT T TAAGTACT CT GT GC T GGAAACAGCACAGAAT CTAC T
TAAACAAGGCAAAAT GCC GT GT T
V2 TATCTCGTCAACTTGTTGGCGAGA (SEQ ID NO: 500)

[00340] In particular, the ability of in vitro single AAV-mediated delivery of gene-editing components to introduce productive editing and precise segmental deletion in the C2C12 mouse myotubes was tested (Table 18B, Figure 10). In Figures 10-12, and 14-17, vector IDs vVT 046, 047, 048, 054, and 052 are shown in Table 18B with their associated sgRNA pairs.
vVT009 has a 7SK2-SluCas9-H1m configuration, with a SluV2 scaffold, and a SV40 NLS on the N-terminus and nucleoplasmin NLS on the C-terminus. vVT053B has the same configuration and sequences as vVT053. The B stands for an A deletion on the backbone of pVT053B (outside of ITRs) that was introduced during cloning. The spacer sequences used in these sgRNA pairs are as follows (Table 18A):
Table 18A:
sgRNA ID Protospacer sequence (a comma separates the two protospacer sequences) E45SL18_E45SL4 TTGTCAGAACAcTGAATGCAAC (SEQ ID NO: 553), cTTGaCGCTGCCCAATGCCATC (SEQ ID NO: 300) E455L21_E455L7 TACAGGAACTCCAGGATGGCAT (SEQ ID NO: 148/306), CTGgCAGAaAGgAgAAAGAGGT (SEQ ID NO: 301) E45SaCas9KKH10_E45 TTGaCGCTGCCCAATGCCATCC (SEQ ID NO: 317), SaCas9KKH20 ACAGATGtCAaTATTCTACAaG (SEQ ID NO: 554)

[00341] Selected AAV
configurations were evaluated for vector genome quantitation (Figure 11) and transgene expression (Figure 12), and immunofluorescence in C2C12 mouse myotubes (Figure 13). Three AAV vectors, vVT046, vVT047, and vVT048, showed an average precise segmental deletion frequency higher than 15% in C2C12 mouse myotubes (Figure 10).
Equivalent vector genome copy number (Figure 11) and Cas9 transgene expression (Figure 12A) were observed between all AAV vectors in C2C12 mouse myotubes. Higher sgRNA expression was observed with hU6-H1m-75K, hU6c-hU6c and hU6c-751(2 PolIII promoter combinations (Figure 12B). Slightly lower sgRNA expression was observed with 75K2-H1m promoter combination (Figure 12B). sgRNA
located upstream of Cas9 demonstrated overall higher expression in 75K2-H1m promoter combination (Figure 12B). Cas9 with 2xNLS+ and 3xNLS results in superior Cas9 localization to myonuclei (Figure 13).
Table 18B:
Orientatio Configuration NLS1 Endonucleas NLS2 NLS3 sgRNA pair Scaff Vector old ID
= .z> 4 hU6c-Cas9-hU6c SV-40 (+) S1uCas9 NucleoplasmiN/A E45 Slu18/4 V5 vVT046 c-Myc-4 hU6c-Cas9-hU6c S1uCas9 SV-40 -GS GS Nucleoolasmin-E45 S1u18/4 V5 vVT047 GSVD GSGS
c-Myc- = c> 4 hU6c-Cas9-7SK2 S1uCas9 SV-40 -GS GS Nucleoolasmin-E45 S1u18/4 V5 vVT048 GSVD GSGS
c-Myc- = c> 4 H1m-Cas9-M11 S1uCas9 SV-40-GSGS Nucleoplasmin-E45 S1u18/4 V5 yVT049 GSVD GSGS
c-Myc-= c> 4 7SK2-Cas9-H1m S1uCas9 SV-40-GSGS
Nucleoplasmin-E45 S1u18/4 V5 yVT050 GSVD GSGS
= 4 7SK2-Cas9-H1m SV-40 (+) S1uCas9 NucleoplasmiN/A E45 Slu18/4 V5 yVT051 = 4 7SK2-Cas9-H1m SV-40 (+) S1uCas9 NucleoplasmiN/A E45S1u21/7 V5 yVT052 = c> 4 7SK2-Cas9-H1m SV-40 (+) SaCas9-KKH Nucleoplasmi N/A E45SaKKHV2 yVT053 c-Myc-= 4 7SK2-Cas9-H1m S1uCas9 SV-40-GSGS Nucleoplasmin-E45S1u24 V5 yVT054 GSVD GSGS

[00342] The ability of in vivo single AAV-mediated delivery of gene-editing components to successfully reframe exon 45 in the cardiac and skeletal muscle of dEx44 mice was tested. All selected AAV configurations showed an average total indel frequency higher than 12.5% and E45S1u18/4 sgRNA pair showed an average precise segmental deletion frequency higher than 15% in the heart (Figure 14A). vVT047 with E45S1u18/4 sgRNA pair showed an average precise segmental deletion frequency higher than 5% in quadriceps (Figure 14B). All selected AAV
configurations showed an average total dystrophin protein restoration higher than 30% of WT
in hearts of dEx44 DMD mice (Figure 15A). vVT054, vVT046, vVT047 and vVT048 showed an average total dystrophin protein restoration higher than 10% of WT in quadriceps of dEx44 DMD mice (Figure 15B). AAV vectors with 2xNLS+ and 3xNLS enhances editing efficiency and dystrophin restoration in skeletal muscles (Figure 15B). Equivalent AAV vector genome was observed in all groups in heart and skeletal muscles. Higher overall AAV vector copy was observed in heart than compared to skeletal muscle (Figure 16A and 16B). The upstream sgRNA was more efficiently expressed than the downstream cassette in vVT046, vVT047, vVT048 AAV vectors (Figure 17A and 17B). Both upstream and downstream sgRNA expression levels were decreased relative to Cas9 levels in muscle compared to heart tissue (Figure 17A and 17B).

[00343] Similar experiments were performed with a variety of sgRNAs (see spacer sequences in Table 19; Figures 18A-18C) and Cas12i2 endonuclease in exon 45 in HEK293FT
cells.
Table 19:
sgRNA ID Protospacer sequences (a comma separates the two protospacer sequences) E45CAS12I2TTN26 E45CAS TTCCCCAGTTGCATTCAATG (SEQ ID NO: 555), 12I2TTN39 GGCAGCGGCAAACTGTTGTC (SEQ ID NO: 556) E45CAS12I2TTN27 E45CAS CCCAGTTGCATTCAATGTTC (SEQ ID NO: 557), 12I2TTN39 GGCAGCGGCAAACTGTTGTC (SEQ ID NO: 556) E45CAS12I2TTN29 E45CAS AATGTTCTGACAACAGTTTG (SEQ ID NO: 558), 12I2TTN43 TGGTATCTTACAGGAACTCC (SEQ ID NO: 559) E45CAS12I2TTN27 E45CAS CCCAGTTGCATTCAATGTTC (SEQ ID NO: 557), 1212TTN40 CAGGAACTCCAGGATGGCAT (SEQ ID NO: 560) E45CAS12I2TTN19 E45CAS TTGAGGATTGCTGAATTATT(SEQ ID NO: 561), 12I2TTN39 GGCAGCGGCAAACTGTTGTC (SEQ ID NO: 556) E45CAS12I2TTN26 E45CAS TTCCCCAGTTGCATTCAATG (SEQ ID NO:
1212TTN40 555),CAGGAACTCCAGGATGGCAT (SEQ ID NO: 560) E45CAS12I2TTN39 E45CAS GGCAGCGGCAAACTGTTGTC (SEQ ID NO:
12I2TTN43 556),TGGTATCTTACAGGAACTCC (SEQ ID NO: 559) E45CAS12I2TTN19 E45CAS TTGAGGATTGCTGAATTATT (SEQ ID NO: 561), 12I2TTN29 AATGTTCTGACAACAGTTTG (SEQ ID NO: 558) E45CAS12I2TTN17 E45CAS CCTGTAGAATACTGGCATCT (SEQ ID NO: 562), 12I2TTN39 GGCAGCGGCAAACTGTTGTC (SEQ ID NO: 556) E45CAS12I2TTN18 E45CAS CTGTAGAATACTGGCATCTG (SEQ ID NO: 563), 12I2TTN25 CTTCCCCAGTTGCATTCAAT (SEQ ID NO: 564) E45CAS12I2TTN15 E45CAS TTCCTGTAGAATACTGGCAT (SEQ ID NO: 565), 12I2TTN25 CTTCCCCAGTTGCATTCAAT (SEQ ID NO: 564) E45CAS12I2TTN16_E45CAS TCCTGTAGAATACTGGCATC (SEQ ID NO: 566), 12I2TTN26 TTCCCCAGTTGCATTCAATG (SEQ ID NO: 555) E45CAS12I2TTN16 E45CAS TCCTGTAGAATACTGGCATC (SEQ ID NO: 566), 12I2TTN27 CCCAGTTGCATTCAATGTTC (SEQ ID NO: 557) E45CAS1212TTN21 E45CAS GAGGATTGCTGAATTATTTC (SEQ ID NO: 567), 12I2TTN43 TGGTATCTTACAGGAACTCC (SEQ ID NO: 559) E45CAS12I2TTN38 E45CAS TCAGAACATTGAATGCAACT (SEQ ID NO: 568), 12I2TTN43 TGGTATCTTACAGGAACTCC (SEQ ID NO: 559) E45CAS12I2TTN18 E45CAS CTGTAGAATACTGGCATCTG (SEQ ID NO: 563), 12I2TTN28 CATTCAATGTTCTGACAACA (SEQ ID NO: 569) E45CAS1212TTN21 E45CAS GAGGATTGCTGAATTATTTC (SEQ ID NO: 567), 12I2TTN32 CCGCTGCCCAATGCCATCCT (SEQ ID NO: 570) E45CA512I2TTN26_E45CAS TTCCCCAGTTGCATTCAATG (SEQ ID NO: 555), 12I2TTN36 AGCAATCCTCAAAAACAGAT (SEQ ID NO: 571) E45CAS12I2TTN32 E45CAS CCGCTGCCCAATGCCATCCT (SEQ ID NO: 570), 12I2TTN38 TCAGAACATTGAATGCAACT (SEQ ID NO: 568) E45CAS12I2TTN15 E45CAS TTCCTGTAGAATACTGGCAT (SEQ ID NO:
12I2TTN28 565),CATTCAATGTTCTGACAACA (SEQ ID NO: 569) E45CAS12I2TTN27 E45CAS CCCAGTTGCATTCAATGTTC (SEQ ID NO:
12I2TTN36 557),AGCAATCCTCAAAAACAGAT (SEQ ID NO: 571) E45CAS12I2TTN17 E45CAS CCTGTAGAATACTGGCATCT (SEQ ID NO:
12I2TTN29 562),AATGTTCTGACAACAGTTTG (SEQ ID NO: 558) E45CAS12I2TTN18 E45CAS CTGTAGAATACTGGCATCTG (SEQ ID NO:
12I2TTN40 563),CAGGAACTCCAGGATGGCAT (SEQ ID NO: 560) E45CAS12I2TTN15 E45CAS TTCCTGTAGAATACTGGCAT (SEQ ID NO:
12I2TTN40 565),CAGGAACTCCAGGATGGCAT (SEQ ID NO: 560) E45CAS12I2TTN18 E45CAS CTGTAGAATACTGGCATCTG (SEQ ID NO:
12I2TTN30 563),TGACAACAGTTTGCCGCTGC (SEQ ID NO: 572) E45CAS12I2TTN15 E45CAS TTCCTGTAGAATACTGGCAT (SEQ ID NO:
12I2TTN30 565),TGACAACAGTTTGCCGCTGC (SEQ ID NO: 572) E45CAS12I2TTN16 E45CAS TCCTGTAGAATACTGGCATC (SEQ ID NO:
12I2TTN43 566),TGGTATCTTACAGGAACTCC (SEQ ID NO: 559) E45CAS12I2TTN16 E45CAS TCCTGTAGAATACTGGCATC (SEQ ID NO:
12I2TTN32 566),CCGCTGCCCAATGCCATCCT (SEQ ID NO: 570) E45CAS12I2TTN11 E45CAS GCAGACCTCCTGCCACCGCA (SEQ ID NO:
12I2TTN25 573),CTTCCCCAGTTGCATTCAAT (SEQ ID NO: 564) E45CAS12I2TTN12 E45CAS CAGACCTCCTGCCACCGCAG (SEQ ID NO:
12I2TTN26 574),TTCCCCAGTTGCATTCAATG (SEQ ID NO: 555) E45CAS12I2TTN26 E45CAS TTCCCCAGTTGCATTCAATG (SEQ ID NO:
12I2TTN35 555),TACAGGAAAAATTGGGAAGC (SEQ ID NO: 5751 -----E45CAS12I2TTN36_E45CAS AGCAATCCTCAAAAACAGAT (SEQ ID NO:
12I2TTN43 571),TGGTATCTTACAGGAACTCC (SEQ ID NO: 5591 -----E45CAS12I2TTN12 E45CAS CAGACCTCCTGCCACCGCAG (SEQ ID NO:
12I2TTN27 574),CCCAGTTGCATTCAATGTTC (SEQ ID NO: 5571 -----E45CAS12I2TTN27_E45CAS CCCAGTTGCATTCAATGTTC (SEQ ID NO:
12I2TTN35 557),TACAGGAAAAATTGGGAAGC (SEQID NO: 575) E45CAS12I2TTN32 E45CAS CCGCTGCCCAATGCCATCCT (SEQ ID NO:
12I2TTN36 570),AGCAATCCTCAAAAACAGAT (SEQ ID NO: 5711 -----E45CAS12I2TTN11 E45CAS GCAGACCTCCTGCCACCGCA (SEQ ID NO:
12I2TTN28 573),CATTCAATGTTCTGACAACA (SEQ ID NO: 569) E45CAS12I2TTN34 E45CAS GGAAGCCTGAATCTGCGGTG (SEQ ID NO:
12I2TTN39 576),GGCAGCGGCAAACTGTTGTC (SEQ ID NO: 556) E45CAS12I2TTN39_E45CAS GGCAGCGGCAAACTGTTGTC (SEQ ID NO: 556), 12I2TTN7 TTCTGTCTGACAGCTGTTTG (SEQ ID NO: 577) E45CAS12I2TTN11 E45CAS GCAGACCTCCTGCCACCGCA (SEQ ID NO:
12I2TTN40 573),CAGGAACTCCAGGATGGCAT (SEQ ID NO: 560) E45CAS12I2TTN11 E45CAS GCAGACCTCCTGCCACCGCA (SEQ ID NO:
12I2TTN30 573),TGACAACAGTTTGCCGCTGC (SEQ ID NO: 572) E45CAS12I2TTN12 E45CAS CAGACCTCCTGCCACCGCAG (SEQ ID NO:
12I2TTN43 574),TGGTATCTTACAGGAACTCC (SEQ ID NO: 559) E45CAS12I2TTN29_E45CAS AATGTTCTGACAACAGTTTG (SEQ ID NO:
12I2TTN34 558),GGAAGCCTGAATCTGCGGTG (SEQ ID NO: 576) E45CAS12I2TTN35 E45CAS TACAGGAAAAATTGGGAAGC (SEQ ID NO:
12I2TTN43 575),TGGTATCTTACAGGAACTCC (SEQ ID NO: 559) E45CAS12I2TTN29_E45CAS AATGTTCTGACAACAGTTTG (SEQ ID NO:
12I2TTN7 558),TTCTGTCTGACAGCTGTTTG (SEQ ID NO: 577) E45CAS12I2TTN12 E45CAS CAGACCTCCTGCCACCGCAG (SEQ ID NO:
12I2TTN32 574),CCGCTGCCCAATGCCATCCT (SEQ ID NO: 570) E45CAS12I2TTN32 E45CAS CCGCTGCCCAATGCCATCCT (SEQ ID NO:
12I2TTN35 570),TACAGGAAAAATTGGGAAGC (SEQ ID NO: 575)

[00344] This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about,"
to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[00345] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

Claims

What is claimed is:
1. A composition comprising:
a. a single nucleic acid molecule comprising:
i. a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; or ii. a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii. a nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and 1, 2, or 3 guide RNAs; or b. two nucleic acid molecules comprising:
i. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9); and a second nucleic acid that does not encode a SaCas9 or SluCas9 and encodes any one of the following:
1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or 2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid;
or 3. from one to six guide RNAs; or ii. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9) and 1. at least one, at least two, or at least three guide RNAs; or 2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid;
or 3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SaCas9 or S1uCas9, optionally wherein the second nucleic acid comprises any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or 2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid;
or 3. from one to six guide RNAs; or iii. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (S1uCas9) and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SaCas9 or S1uCas9 and encodes from one to six guide RNAs; or iv. a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (S1uCas9) and at least two guide RNAs, wherein at least one guide RNA binds upstream of a target sequence and at least one guide RNA binds downstream of the target sequence; and a second nucleic acid that does not encode a SaCas9 or S1uCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid, wherein the guide RNA(s) target a region in the dystrophin gene.

2. A composition comprising two nucleic acid molecules comprising i) a first nucleic acid encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (S1uCas9) and a first and a second guide RNA that function to excise a portion of a DMD
gene; and ii) a second nucleic acid encoding at least 2 or at least 3 copies of the first guide RNA and at least 2 or at least 3 copies of the second guide RNA.

3. A composition comprising one or more nucleic acid molecules encoding an endonuclease and a pair of guide RNAs, wherein each guide RNA targets a different sequence in a DMD gene, wherein the endonuclease and pair of guide RNAs are capable of excising a DNA
fragment from the DMD gene; wherein the DNA fragment is between 5-250 nucleotides in length.

4. The composition of claim 3, wherein the endonuclease is a class 2, type II Cas endonuclease.

5. The composition of claim 3, wherein the class 2, type II Cas endonuclease is SpCas9, SaCas9, or S1uCas9.

6. The composition of claim 3, wherein the endonuclease is not a class 2, type V Cas endonuclease.

7. The composition of claim 3, wherein the excised DNA fragment comprises a splice acceptor site or a splice donor site.

8. The composition of claim 3, wherein the excised DNA fragment comprises a premature stop codon in the DMD gene.

9. The composition of claim 3, wherein the excised DNA fragment does not comprise an entire exon of the DMD gene.

10. The composition of any one of claims 1-9, wherein the guide RNA comprises any one of the following:
a. when SaCas9 is used, one or more spacer sequences selected from any one of SEQ ID
NOs: 1-35, 1000-1078, and 3000-3069; or b. when S1uCas9a is used, one or more spacer sequences selected from any one of SEQ
ID NOs: 100-225, 2000-2116, and 4000-4251; or c. when SaCas9 is used, one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:
1-35, 1000-1078, and 3000-3069; or d. when S1uCas9a is used, one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:
100-225, 2000-2116, and 4000-4251; or e. when SaCas9 is used, one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 1-35, 1000-1078, and 3000-3069; or f. when S1uCas9 is used, one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 100-225, 2000-2116, and 4000-4251; or g. when SaCas9 is used with at least two guide RNAs, a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ
ID
NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16;

1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016;
1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12;
1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; or h. when SaCas9 is used with at least two guide RNAs, at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10;
1005 and 1017; 16 and 1017; and 16 and 1018; or i. when SaCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10;
16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16;
1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017;
and 16 and 1018; or j. when S1uCas9 is used with at least two guide RNAs, a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ
ID
NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or k. when S1uCas9 is used with at least two guide RNAs, at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135;

150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131;

and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or 1. when S1uCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or m. when S1uCas9 is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150; and v. SEQ ID Nos: 146 and 148; or n. when a SaCas9-KKH is used with at least two guide RNAs, at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016.

11. A composition comprising a single nucleic acid molecule encoding one or more guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises:
a. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or b. a first nucleic acid encoding one or more spacer sequences selected from any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9); or c. a first nucleic acid encoding one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:
1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or d. a first nucleic acid encoding one or more spacer sequences comprising at least 20 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID
NOs:

100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9); or e. a first nucleic acid encoding one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or f. a first nucleic acid encoding one or more spacer sequences that is at least 90%
identical to any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 and a second nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9); or g. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12;
1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or h. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16;
12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003;
12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16;
1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or i. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005;
1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012;
12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10;

and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003;

and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and a second nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9); or j. a first nucleic acid encoding a pair of guide RNAs comprising a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136;
139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149;

and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9); or k. a first nucleic acid encoding a pair of guide RNAs comprising at least 17, 18, 19, 20, or 21 contiguous nucleotides of a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9); or 1. a first nucleic acid encoding a pair of guide RNAs that is at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences: SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and a second nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9); or m. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150;
v. SEQ ID Nos: 146 and 148;

and a second nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9); or n. a first nucleic acid encoding a pair of guide RNAs that is at least 90%
identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016;
and a second nucleic acid encoding a SaCas9-KKH.

12. A composition comprising one or more nucleic acid molecules encoding a Staphylococcus lugdunensis (S1uCas9) and at least two guide RNAs, wherein a first and second guide RNA
target different sequences in a DMD gene, wherein the first and a second guide RNA
comprise a sequence that is at least 90% identical to a first and second spacer sequence selected from any one of the following pairs of spacer sequences:
i. SEQ ID NOS: 148 and 134, SEQ ID Nos: 145 and 131, SEQ ID Nos: 144 and 149;
iv. SEQ ID Nos: 144 and 150;
v. SEQ ID Nos: 146 and 148.

13. A composition comprising one or more nucleic acid molecules encoding an endonuclease and at least two guide RNAs, wherein the guide RNAs each target a different sequence in a DMD
gene, wherein the guide RNAs each comprise a sequence that is at least 90%
identical to a first and second spacer sequence selected from any one of the following pairs of spacer se quence s:
i. SEQ ID NOs: 12 and 1013; and SEQ ID Nos: 12 and 1016; and a second nucleic acid encoding a SaCas9-KKH.

14. The composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least two guide RNAs.

15. The composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least three guide RNAs.

16. The composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least four guide RNAs.

17. The composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least five guide RNAs.

18. The composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least six guide RNAs.

19. The composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least seven guide RNAs.

20. The composition of any one of the preceding claims, wherein the first and/or the second nucleic acid, if present, encodes at least eight guide RNAs.

21. The composition of any one of the preceding claims, wherein the first nucleic acid comprises a nucleotide sequence encoding an endonuclease and at least one, at least two, or at least three guide RNAs.

22. The composition of any one of the preceding claims, wherein the first nucleic acid comprises a nucleotide sequence encoding an endonuclease and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid.

23. The composition of any one of the preceding claims, wherein the first nucleic acid comprises a nucleotide sequence encoding an endonuclease and from one to three guide RNAs.

24. The composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs.

25. The composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid.

26. The composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes from one to six guide RNAs.

27. The composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 guide RNAs.

28. The composition of any one of the preceding claims, wherein the second nucleic acid, if present, encodes 2, 3, 4, 5, or 6 guide RNAs.

29. The composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the guide RNA encoded by the first nucleic acid and the second nucleic acid are the same.

30. The composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the guide RNA encoded by the first nucleic acid and the second nucleic acid are different.

31. The composition of any one of the preceding claims, comprising at least two nucleic acid molecules encoding at least two guide RNAs, wherein at least one guide RNA
binds to a target sequence within an exon in the DMD gene that is upstream of a premature stop codon, and wherein at least one guide RNA binds to a target sequence within an exon in the DMD
gene that is downstream of a premature stop codon.

32. The composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the first nucleic acid molecule and the second nucleic acid molecule each encode the same guide RNA.

33. The composition of any one of the preceding claims, comprising at least two nucleic acid molecules each encoding at least one guide RNA, wherein the second nucleic acid molecule encodes a guide RNA that binds to the same target sequence as the guide RNA in the first nucleic acid molecule.

34. The composition of any one of the preceding claims, comprising at least two nucleic acid molecules, wherein the second nucleic acid molecule encodes at least 2, at least 3, at least 4, at least 5, or at least 6 guide RNAs, wherein the guide RNAs in the second nucleic acid molecule bind to the same target sequence as the guide RNA in the first nucleic acid molecule.

35. The composition of any one of the preceding claims, wherein the composition comprises at least two nucleic acid molecules, wherein the first nucleic acid molecule comprises a sequence encoding an endonuclease, wherein the second nucleic acid molecule encodes a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA
are not the same sequence, and wherein the second nucleic acid molecule does not encode an endonuclease.

36. The composition of claim 35, wherein the first nucleic acid molecule also encodes a copy of the first guide RNA and a copy of the second guide RNA.

37. The composition of claim 35 or 36, wherein the second nucleic acid molecule encodes two copies of the first guide RNA and two copies of the second guide RNA.

38. The composition of any one of claims 35-37, wherein the second nucleic acid molecule encodes three copies of the first guide RNA and three copies of the second guide RNA.

39. The composition of any one of claims 35-37, wherein the first nucleic acid molecule comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence, the reverse complement of a nucleotide sequence encoding the first guide RNA sequence, the reverse complement of a promoter for expression of the nucleotide sequence encoding the first guide RNA sequence, a promoter for expression of a nucleotide sequence encoding the endonuclease, a nucleotide sequence encoding an endonuclease, a polyadenylation sequence, a promoter for expression of the second guide RNA in the same direction as the promoter for the endonuclease, the second guide RNA
sequence, and a second guide RNA scaffold sequence.

40. The composition of claim 39, wherein the promoter for expression of the nucleotide sequence encoding the first guide RNA sequence in the first nucleic acid molecule is a U6 promoter and the promoter for expression of the nucleotide sequence encoding the second guide RNA
in the first nucleic acid molecule is a U6 promoter.

41. The composition of any one of claims 35-40, wherein the first nucleic acid molecule is in a first vector, and wherein the second nucleic acid is in a separate second vector.

42. The composition of any one of claims 35-41, wherein the first nucleic acid molecule encodes a Staphylococcus aureus Cas9 (SaCas9) endonuclease, and wherein the first guide RNA
comprises the first sequence and the second guide RNAs comprises the second sequence from any one the following pairs of sequences: SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;
1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003;
1003 and 16;
1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018.

43. The composition of claim 42, wherein the first guide RNA comprises the sequence of SEQ ID
NO: 12 and the second guide RNA comprises the sequence of SEQ ID NO: 1013.

44. The composition of any one of claims 35-43, wherein the first nucleic acid molecule encodes a Staphylococcus lugdunensis (S1uCas9) endonuclease, and wherein the first guide RNA
comprises the first sequence and the second guide RNAs comprises the second sequence from any one the following pairs of sequences: SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148;
135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139;
131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146.

45. The composition of claim 44, wherein:
a) the first guide RNA comprises the sequence of SEQ ID NO: 148 and the second guide RNA comprises the sequence of SEQ ID NO: 134; or b) the second guide RNA comprises the sequence of SEQ ID NO: 145 and the second guide RNA comprises the sequence of SEQ ID NO: 131.

46. The composition of any one of claims 35-45, wherein the first nucleic acid is in a first vector, and wherein the second nucleic acid is in a separate second vector.

47. The composition of claim 46, wherein the first and second vectors are viral vectors.

48. The composition of claim 47, wherein the viral vectors are AAV9 vectors.

49. The composition of claim 48, wherein the AAV9 vectors are each less than 5kb, less than 4.9kb, less than 4.85, less than 4.8, or less than 4.75 kb in size from ITR to ITR, inclusive of both ITRs.

50. The composition of claim 48 or 49, wherein the AAV9 vectors are each between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs.

51. The composition of any one of claims 47-50, wherein the first vector is between 4.4 - 4.85 kb from ITR to ITR in size, inclusive of both ITRs.

52. The composition of any one of the preceding claims, wherein the guide RNA
binds to one or more target sequences within a DMD gene.

53. The composition of any one of the preceding claims, wherein the guide RNA
binds to one or more target sequences within an exon of the DMD gene.

54. The composition of any one of the preceding claims, comprising two guide RNAs, wherein i) each guide RNA targets a sequence within an exon; ii) one guide RNA targets a sequence within an exon and one targets a sequence within an intron; or iii) each guide RNA targets a sequence within an intron.

55. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein i) each guide RNA targets the same genomic target sequence; ii) each guide RNA
targets a different target sequence; or iii) at least one guide RNA targets one sequence and at least one guide RNA targets a different sequence.

56. The composition of any one of the preceding claims, comprising a guide RNA
that binds to an exon of the DMD gene, wherein the exon is selected from exon 43, 44, 45, 50, 51, and 53.

57. The composition of any one of the preceding claims, comprising at least two guide RNAs that binds to an exon of the DMD gene, wherein at least one guide RNA binds to a target sequence within an exon in the DMD gene, and at least one guide RNA binds to a different target sequence within the same exon in the DMD gene.

58. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein at least one guide RNA binds to a target sequence within an exon that is upstream of a premature stop codon, and at least one guide RNA binds to a target sequence within an exon that is downstream of a premature stop codon.

59. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein at least one guide RNA binds to a target sequence within an exon in the DMD gene, and at least one guide RNA binds to a different target sequence within the same exon in the DMD gene, wherein when expressed in vivo or in vivo, a portion of the exon is excised.

60. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon.

61. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, and wherein the portion of the exon remaining after excision are rejoined with a one nucleotide insertion.

62. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, wherein the portion of the exon remaining after excision is rejoined without a nucleotide insertion.

63. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, wherein the size of the excised portion of the exon is between 5 and 250 nucleotides in length.

64. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, wherein the size of the excised portion of the exon is between 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 75, 5 and 50, 5 and 25, 5 and 10, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 75, 20 and 50, 20 and 25, 50 and 250, 50 and 200, 50 and 150, 50 and 100, and 50 and 75 nucleotides.

65. The composition of any one of the preceding claims, comprising at least two guide RNAs, wherein once expressed in vitro or in vivo in combination with an RNA-guided endonuclease, the guide RNAs excise a portion of the exon, wherein the size of the excised portion of the exon is between 8 and 167 nucleotides.

66. The composition of any one of the preceding claims, wherein the guide RNA
is an sgRNA.

67. The composition of any one of the preceding claims, wherein the guide RNA
is modified.

68. The composition of any one of the preceding claims, wherein the guide RNA
is modified, and wherein the modification alters one or more 2' positions and/or phosphodiester linkages.

69. The composition of any one of the preceding claims, wherein the guide RNA
is modified, and wherein the modification alters one or more, or all, of the first three nucleotides of the guide RNA and/or the last three nucleotides of the sgRNA.

70. The composition of any one of claims 66-69, wherein the modification alters one or more, or all, of the last three nucleotides of the guide RNA.

71. The composition of any one of the preceding claims, wherein the guide RNA
is modified, and wherein the modification includes one or more of a phosphorothioate modification, a 2'-0Me modification, a 2'-0-MOE modification, a 2' -F modification, a 2'-0-methine-4' bridge modification, a 3'-thiophosphonoacetate modification, or a 2'-deoxy modification.

72. The composition of any one of the preceding claims, wherein the composition further comprises a pharmaceutically acceptable excipient.

73. The composition of any one of the preceding claims, wherein the composition is associated with a lipid nanoparticle (LNP).

74. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector.

75. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, and wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector.

76. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, and wherein the viral vector is an adeno-associated virus (AAV) vector.

77. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10, AAVrh74, or AAV9 vector, wherein the number following AAV indicates the AAV serotype.

78. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAV serotype 9 (AAV9) vector.

79. The composition of claim 78, wherein the AAV serotype 9 vector is less than 5kb, less than 4.9kb, less than 4.85, less than 4.8, or less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs.

80. The composition of claim 78 or 79, wherein the AAV serotype 9 vector is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs.

81. The composition of any one of claims 78-80, wherein the AAV serotype 9 vector is between 4.4-4.85 kb from ITR to ITR in size, inclusive of both ITRs.

82. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAVrh10 vector.

83. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and wherein the AAV vector is an AAVrh74 vector.

84. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector comprises a tissue-specific promoter.

85. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector comprises a muscle-specific promoter, optionally wherein the muscle-specific promoter is a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, an SPc5-12 promoter, or a CK8e promoter.

86. The composition of any one of the preceding claims, wherein the composition is associated with a viral vector, wherein the viral vector comprises any one or more of the following promoters: U6, H1, and 7SK promoter.

87. The composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein at least one guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 12, 15, 16, 20, 27, 28, 32, 33, and 35.

88. The composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ ID NO:
711.

89. The composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ ID
NO: 711.

90. The composition of any one of the preceding claims, comprising a nucleic acid encoding SaCas9, wherein the SaCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 715-717.

91. The composition of any one of the preceding claims, comprising a nucleic acid encoding SluCas9, wherein at least one guide RNA comprises a spacer sequence selected from any one of SEQ ID NOs: 131, 134, 135, 136 ,139, 144, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, and 225.

92. The composition of any one of the preceding claims, comprising a nucleic acid encoding S1uCas9, wherein the SluCas9 comprises the amino acid sequence of SEQ ID NO:
712.

93. The composition of any one of the preceding claims, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 is a variant of the amino acid sequence of SEQ ID
NO: 712.

94. The composition of any one of the preceding claims, comprising a nucleic acid encoding SluCas9, wherein the SluCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 718-720.

95. The composition of claim 1, wherein the single nucleic acid molecule or the first nucleic acid comprises from 5' to 3' with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first guide RNA scaffold sequence, the reverse complement of a nucleotide sequence encoding the first guide RNA sequence, the reverse complement of a promoter for expression of the nucleotide sequence encoding the first guide RNA sequence, a promoter for expression of a nucleotide sequence encoding Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9), a nucleotide sequence encoding the SaCas9 or SluCas9, a polyadenylation sequence, a promoter for expression of the second guide RNA in the same direction as the promoter for the SaCas9 or SluCas9, a nucleotide sequence encoding the second guide RNA
sequence, and a nucleotide sequence encoding the second guide RNA scaffold sequence.

96. The composition of claim 95, wherein the promoter for expression of the nucleic acid encoding the first guide RNA sequence is a U6 promoter and the promoter for expression of the nucleic acid encoding the second guide RNA is a U6 promoter.

97. The composition of claim 95 or 96, wherein the SaCas9 or SluCas9 comprise at least two nuclear localization signals (NLSs).

98. The composition of claim 97, wherein the SaCas9 or S1uCas9 comprise a c-Myc NLS fused to the N-terminus of the SaCas9 or S1uCas9, optionally by means of a linker.

99. The composition of claim 97 or 98, wherein the SaCas9 or S1uCas9 comprise an 5V40 NLS
fused to the C-terminus of the SaCas9 or S1uCas9, optionally by means of a linker.

100. The composition of any one of claims 95-99, wherein the SaCas9 or S1uCas9 comprise a nucleoplasmin NLS fused to the C-terminus of the SaCas9 or S1uCas9, optionally by means of a linker.

101. The composition of any one of claims 95-100, wherein the SaCas9 or S1uCas9 comprise:
a) a c-Myc NLS fused to the N-terminus of the SaCas9 or S1uCas9, optionally by means of a linker, b) an 5V40 NLS fused to the C-terminus of the SaCas9 or S1uCas9, optionally by means of a linker, and c) a nucleoplasmin NLS fused to the C-terminus of the 5V40 NLS, optionally by means of a linker,

102. The composition of any one of claims 95-101, wherein the scaffold sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901.

103. The composition of any one of claims 95-102, wherein the scaffold sequence for the second guide RNA comprises the sequence of SEQ ID NO: 901.

104. The composition of any one claims 95-103, wherein the single nucleic acid molecule or the first nucleic acid is less than 5kb, less than 4.9kb, 4.85, 4.8, or 4.75 kb.

105. The composition of any one of claims 95-104, wherein the single nucleic acid molecule or the first nucleic acid is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb.

106. The composition of claim 105, wherein the single nucleic acid molecule or the first nucleic acid is between 4.4-4.85 kb.

107. A composition comprising a guide RNA encoded by a sequence comprising any one of SEQ ID NOs: 1-35, 1000-1078, or 3000-3069 or complements thereof

108. A composition comprising a guide RNA encoded by a sequence comprising any one of SEQ ID NOs: 100-225, 2000-2116, or 4000-4251 or complements thereof

109. The composition of any one of claims 1-108 for use in treating Duchenne Muscular Dystrophy (DMD).

110. The composition of any one of claims 1-108 for use in making one or more double strand breaks in any one or more of exons 43, 44, 45, 50, 51, or 53 of the dystrophin gene.

111. A method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell the composition of any one of claims 1-108.

112. A method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:
a. a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, or 3000-3069;
b. a spacer sequence comprising at least 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 1-35, 1000-1078, or 3000-3069; or c. a spacer sequence that is at least 90% identical to any one of SEQ ID
NOs: 1-35, 1000-1078, 3000-3069; and ii) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).

113. A method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid molecule encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:
a. a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, or 4000-4251;
b. a spacer sequence comprising at least 20 contiguous nucleotides of a spacer sequence selected from SEQ ID NOs: 100-225, 2000-2116, or 4000-4251; or c. a spacer sequence that is at least 90% identical to any one of SEQ ID NOs:

225, 2000-2116, or 4000-4251; and ii) a nucleic acid molecule encoding Staphylococcus lugdunensis (S1uCas9).

114. A method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding a pair of guide RNAs comprising:
a. a first and second spacer sequence selected from any one of SEQ ID
NOs: 10 and 15;
and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005;
1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018;

b. a first and second spacer sequence comprising at least 17, 18, 19, 20, or contiguous nucleotides of any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16;
1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001;
1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10;
1005 and 1017; 16 and 1017; and 16 and 1018; or c. a first and second spacer sequence that is at least 90% identical to any one of SEQ ID
NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16;

1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016;
1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12;
1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018; and ii) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9).

115. A method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding a pair of guide RNAs comprising:
a. a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; and 146 and 148;
b. a first and second spacer sequence comprising at least 17, 18, 19, 20, or contiguous nucleotides of any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; or c. a first and second spacer sequence that is at least 90% identical to any one of SEQ ID
NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146; and ii) a nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9).

116. The method of any one of the previous method or use claims, wherein the single nucleic acid molecule is delivered to the cell on a single vector.

117. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the spacer sequence is selected from any one of SEQ ID
NOs: 12, 15, 16, 20, 27, 28, 32, 33, and 35.

118. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ ID
NO: 711.

119. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 is a variant of the amino acid sequence of SEQ
ID NO: 711.

120. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding SaCas9, wherein the SaCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 715-717.

121. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding S1uCas9, wherein the spacer sequence is selected from any one of SEQ ID
NOs: 131, 134, 135, 136 ,139, 144, 148, 149, 150, 151, 179, 184, 201, 210, 223, 224, and 225.

122. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding S1uCas9, wherein the S1uCas9 comprises the amino acid sequence of SEQ ID
NO: 712.

123. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding S1uCas9, wherein the S1uCas9 is a variant of the amino acid sequence of SEQ
ID NO: 712.

124. The method of any one of the previous method or use claims, comprising a nucleic acid molecule encoding S1uCas9, wherein the S1uCas9 comprises an amino acid sequence selected from any one of SEQ ID NOs: 718-720.

125. A method of excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:

i) a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA
binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA;
and ii) a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9);
wherein the pair of guide RNAs and SaCas9 excise a portion of the exon.

126. A method of excising a portion of an exon with a premature stop codon in a subject with Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell a single nucleic acid molecule comprising:
i) a nucleic acid encoding a pair of guide RNAs wherein the first guide RNA
binds to a target sequence within the exon that is upstream of the premature stop codon, and wherein the second guide RNA binds to a sequence that is downstream of the premature stop codon and downstream of the sequence bound by the first guide RNA;
and ii) a nucleic acid encoding a Staphylococcus lugdunensis (S1uCas9);
wherein the pair of guide RNAs and SaCas9 excise a portion of the exon.

127. The method of any one of the previous method claims, comprising a single nucleic acid molecule, wherein the single nucleic acid molecule is delivered to the cell on a single vector.

128. The method of any one of the previous method claims, wherein a portion of a DMD
gene is excised, and wherein the portions of the gene remaining after excision are rejoined with a one-nucleotide insertion.

129. The method of any one of the previous method claims, wherein a portion of a DMD
gene is excised, and wherein the portions of the exon remaining after excision are rejoined without a nucleotide insertion.

130. The method of any one of the previous method claims, wherein a portion of a DMD
gene is excised, wherein the size of the excised portion of the gene is between 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 75, 5 and 50, 5 and 25, 5 and 10, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 75, 20 and 50, 20 and 25, 50 and 250, 50 and 200, 50 and 150, 50 and 100, and 50 and 75 nucleotides.

131. The method of any one of the previous method claims, wherein a portion of a DMD
gene is excised, wherein the size of the excised portion of the exon is between 8 and 167 nucleotides.

132. The method of any one of the previous method claims, wherein a portion of a DMD
gene is excised, wherein the portion is within exon 43, 44, 45, 50, 51, or 53.

133. The method of any one of the previous method claims, comprising a pair of guide RNAs, wherein the pair of guide RNA comprise a first and second spacer sequence selected from any one of SEQ ID NOs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16;
1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005;
1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018.

134. The method of any one of the previous method claims, comprising a pair of guide RNAs, wherein the pair of guide RNAs comprise a first and second spacer sequence selected from any one of SEQ ID NOs: 148 and 134; 149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131; 139 and 151; 140 and 131; 140 and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131;
145 and 151; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131;
136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146.

135. The method of any one of the previous method claims, comprising SaCas9, wherein the SaCas9 comprises the amino acid sequence of SEQ ID NO: 715.

136. The composition or method of any one of the preceding claims, wherein the single nucleic acid molecule is an AAV vector, wherein the vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding a SaCas9, a polyadenylation sequence, a promoter for expression of a second sgRNA in the same direction as the promoter for SaCas9, a second sgRNA guide sequence, and a second sgRNA scaffold sequence.

137. The composition or method of claim 136, wherein the promoter for expression of the first sgRNA guide sequence is an hU6 promoter.

138. The composition or method of any one of claims 136-137, wherein the promoter for expression of the second sgRNA guide sequence is an hU6 promoter.

139. The composition or method of any one of claims 136-137, wherein the promoter for the expression of the first sgRNA guide sequence is an hU6 promoter, and the promoter for the expression of the second sgRNA guide sequence is an hU6 promoter.

140. The composition or method of any one of claims 136-137, wherein the promoter for expression of the nucleic acid encoding the first sgRNA guide sequence is an 7SK promoter.

141. The composition or method of any one of claims 136-137, wherein the promoter for expression of the nucleic acid encoding the second sgRNA guide sequence is an 7SK promoter.

142. The composition or method of any one of claims 136-137, wherein the promoter for expression of the nucleic acid encoding the second sgRNA guide sequence is an Hlm promoter.

143. The composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or S1uCas9 is fused to a nucleic acid sequence encoding one or more nuclear localization sequences (NLSs).

144. The composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or S1uCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS).

145. The composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or S1uCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS).

146. The composition or method of any one of the preceding claims, wherein the nucleic acid sequence encoding SaCas9 or S1uCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS).

147. The composition or method of any one of claims 143-146, wherein the one or more NLSs comprises an 5V40 NLS.

148. The composition or method of any one of claims 143-147, wherein the one or more NLSs comprises an c-Myc NLS.

149. The composition or method of any one of claims 143-148, wherein the one or more NLSs comprises a nucleoplasmin NLS.

150. The composition or method of any one of the preceding claims, wherein the nucleic acid encoding the guide RNA or the nucleic acid encoding the pair of guide RNAs comprises a sequence selected from any one of SEQ ID NOs: 600, 601, or 900-917, and wherein the composition or method comprises a nucleic acid encoding S1uCas9.

151. The composition or method of any one of the preceding claims, wherein the nucleic acid encoding the guide RNA or the nucleic acid encoding the pair of guide RNAs comprises a sequence selected from any one of SEQ ID NOs: 901-917, and wherein the composition or method comprises a nucleic acid encoding S1uCas9.

152. The composition of any one of the preceding claims, wherein the nucleic acid molecule encodes at least a first guide RNA and a second guide RNA.

153. The composition of claim 152, wherein the nucleic acid molecule encodes a spacer sequence for the first guide RNA, a scaffold sequence for the first guide RNA, a spacer sequence for the second guide RNA, and a scaffold sequence for the second guide RNA.

154. The composition of claim 152, wherein the spacer sequence for the first guide RNA
and the spacer sequence for the second guide RNA are the same.

155. The composition of claim 152, wherein the spacer sequence for the first guide RNA
and the spacer sequence for the second guide RNA are different.

156. The composition of any one of claims 153-155, wherein the scaffold sequence for the first guide RNA and the scaffold sequence for the second guide RNA are the same.

157. The composition of any one of claims 153-156, wherein the scaffold sequence for the first guide RNA and the scaffold sequence for the second guide RNA are different.

158. The composition of claim 156, wherein the scaffold sequence for the first guide RNA
comprises a sequence selected from the group consisting of SEQ ID NOs: 901-916, and wherein the scaffold sequence for the second guide RNA comprises a different sequence selected from the group consisting of SEQ ID NOs: 901-916.