TW202521691A - Engineered type v rna programmable endonucleases and their uses - Google Patents

Abstract

The present disclosure provides engineered Type V endonucleases suitable for editing eukaryotic genomic DNA, as well as methods of producing the nucleases, systems comprising the nucleases, as well as methods of using the nucleases and systems to edit eukaryotic genomic DNA.

Description

Engineered V-type RNA programmable endonuclease and its use

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes (collectively referred to as CRISPR-Cas or CRISPR/Cas systems) are currently understood to provide immunity to bacteria and archaea against phage infection. The CRISPR-Cas system of prokaryotic adaptive immunity is an extremely diverse group of protein effectors, noncoding elements, and locus architectures, some of which have been engineered and applied to produce important biotechnological applications.

The components of the system involved in host defense include one or more effector proteins capable of modifying DNA or RNA and an RNA guide element responsible for targeting the activity of these proteins to specific sequences on the phage DNA or RNA. The RNA guide consists of CRISPR RNA (crRNA) and may require an additional trans-acting RNA (tracrRNA) to achieve targeted nucleic acid manipulation by the effector protein. The crRNA consists of a segment called a "direct repeat" that is responsible for binding the crRNA to the effector protein and a segment called a "spacer sequence" that is complementary to the desired nucleic acid target sequence. The CRISPR system can be reprogrammed to target alternative DNA or RNA targets by modifying the spacer sequence of the crRNA.

CRISPR-Cas systems can be broadly divided into two categories: Class 1 systems consist of multiple effector proteins complexed together around crRNA, and Class 2 systems consist of a single effector protein complexed with a crRNA guide for targeting DNA or RNA substrates. The single subunit effector components of Class 2 systems provide a simpler set of components for engineering and application and have become an important source of programmable effectors to date. Therefore, the discovery, engineering, and optimization of novel Class 2 systems can lead to broad and powerful programmable technologies for genome engineering and other fields.

In recent years, the RNA-guided DNA-targeting principle of genome editing using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) has been widely developed. Five types of CRISPR-Cas systems have been described (type I, type II and IIb, type III, type V and type VI). Most uses of CRISPR-Cas for genome editing utilize type II systems. The main advantage offered by bacterial type II CRISPR-Cas systems is the minimal requirement for programmable DNA interference: the endonuclease Cas9 guided by a customizable dual RNA structure. As originally demonstrated in the original type II system of Streptococcus pyogenes , transactivating CRISPR RNA (tracrRNA) binds to the invariant repeats of precursor CRISPR RNA (pre-crRNA), thereby forming a duplex-RNA that is essential for RNase III-mediated co-maturation of crRNA in the presence of Cas9 and for Cas9-mediated cleavage of invading DNA. As demonstrated in Streptococcus pyogenes, Cas9 guided by the duplex formed by mature activating tracrRNA and targeting crRNA introduces site-specific double-stranded DNA (dsDNA) breaks in invading homologous DNA. Cas9 is a multi-domain enzyme that uses the HNH nuclease domain to cleave the target strand (defined as complementary to the crRNA spacer sequence) and the RuvC-like domain to cleave the non-target strand.

In addition to the type II CRISPR Cas 9 nuclease, a variety of different type V endonuclease nucleases have been described, such as Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b (Koonin et al., Curr Opin Microbiol. 2017 Jun; 37: 67–78, and Makarova et al., Nat Rev Microbiol. 2020 Feb; 18(2): 67-83.). Some of these systems do not require tracr RNA (Cas 12a, Cas 13a, Cas 13b), while Cas 12b nucleases generally require tracr RNA (Koonin et al., Curr Opin Microbiol. 2017 Jun; 37: 67–78).

WO2022258753A1 discloses novel V-type endonuclease polypeptides called B-GEn.1, B-GEn.1.2 and B-GEn.2, which have particularly advantageous characteristics for eukaryotic genome editing compared to other endonucleases. The present invention provides engineered V-type endonuclease polypeptides with improved gene editing efficiency.

The present invention relates to engineered V-type endonucleases with improved gene editing efficiency. Without being bound by theory, it is believed that the engineered V-type endonucleases have improved gene editing efficiency through better target interaction through their oligonucleotide binding domains (OBDs) (e.g., OBD-II).

The present invention provides engineered V-type endonucleases comprising an amino acid other than aspartic acid at the position of D504 of the V-type endonuclease corresponding to SEQ ID NO: 1 (B-GEn.1) or D501 of the V-type endonuclease of SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2). In some embodiments, the amino acid at the position of D504 of the V-type endonuclease corresponding to SEQ ID NO: 1 (B-GEn.1) or D501 of the V-type endonuclease of SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2) is arginine.

The present invention also provides an engineered V-type nuclease containing an OBD ( _e.g. , OBD-II) comprising a target interaction sequence motif _{GX1X2X3X4NX5X6X7DX8 ( SEQ} ID _NO : 204), wherein each _{of X1} to _X8 is any _amino acid. In an exemplary embodiment, the target interaction sequence motif is any one _of SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 3.

Exemplary engineered V-type endonucleases include B-GEn polypeptides. Engineered V-type endonucleases and B-GEn polypeptides include those described in Chapter 6.2 (and optionally comprising a nuclear localization signal as described in Chapter 6.3 and/or a linker sequence as described in Chapter 6.4) and numbered Examples 1 to 52.

The present invention further provides an engineered V-type endonuclease system, such as an engineered B-GEn V-type endonuclease system, which comprises an engineered B-GEn polypeptide and a suitable guide RNA and/or a nucleic acid encoding it. An exemplary engineered V-type endonuclease system is disclosed in Chapter 6.5 and an exemplary guide RNA is disclosed in Chapter 6.6. In some embodiments, the engineered V-type endonuclease system is a ribonucleoprotein (RNP) complex, which comprises an engineered V-type endonuclease or B-GEn polypeptide and a guide RNA. The ribonucleoprotein complex is described in Chapter 6.7 and in numbered embodiments 72 to 77.

The present invention further provides nucleic acids encoding engineered V-type endonucleases and B-GEn polypeptides, for example, expression vectors for engineered V-type endonucleases and B-GEn polypeptides, and recombinant cells engineered to express engineered V-type endonucleases and B-GEn polypeptides. Exemplary nucleic acids are disclosed in Chapter 6.8 and Examples 53 to 62, exemplary recombinant cells and their use for producing engineered V-type endonucleases and B-GEn polypeptides are described in Chapter 6.10 and Examples 63 to 69, and exemplary vectors are disclosed in Chapter 6.9.

In certain aspects, a method for targeting, editing, modifying or manipulating a target DNA in a cell or in vitro is provided herein. These methods generally require that an engineered V-type endonuclease or B-GEn polypeptide system be introduced into the cell or in vitro environment under conditions suitable for the engineered V-type endonuclease or B-GEn polypeptide to perform one or more nicks or cuts or base editing in the target DNA, wherein the engineered V-type endonucleases or B-GEn polypeptides are guided to the target DNA by a guide RNA in a treated or untreated form. As used herein, the term "V-type endonuclease or B-GEn polypeptide system" is intended to refer to any combination of nucleic acid and polypeptide components that can be delivered to a cell so that an RNP comprising a V-type endonuclease or B-GEn polypeptide can be operably constituted in the cell so that editing can be performed. Therefore, a V-type endonuclease or B-GEn polypeptide system can include any combination of: (a) (i) a V-type endonuclease or B-GEn polypeptide and/or (ii) one or more nucleic acids comprising a nucleotide sequence encoding a V-type endonuclease or B-GEn polypeptide and (b) (i) a guide RNA and/or (ii) a nucleic acid comprising a nucleotide sequence encoding a guide RNA.

In some embodiments, the RNP of the present invention (comprising an engineered V-type endonuclease or B-GEn polypeptide and a guide RNA) is used to edit the genome of a cell. In some embodiments, the method of using RNP for genomic DNA editing comprises nucleofecting a target cell comprising the genomic DNA with the RNP and exposing the target cell to conditions for gene editing to occur, for example, by culturing the target cell under conditions suitable for genome editing by an engineered V-type endonuclease or B-GEn polypeptide.

In some embodiments, one or more viruses (e.g., one or more adeno-associated viruses (AAV)) are used to deliver an engineered V-type endonuclease or B-GEn polypeptide system (e.g., one or more viruses comprising one or more nucleic acids encoding an engineered V-type endonuclease or B-GEn polypeptide and one or more nucleic acids encoding a guide RNA) to a cell so that its genome can be edited. In some embodiments, methods of using one or more viruses to perform genomic DNA editing include contacting a target cell comprising the genomic DNA with the one or more viruses and exposing the target cell to conditions where genetic editing occurs, such as by culturing the target cell under conditions suitable for expression of the engineered V-type nuclease or B-GEn polypeptide and guide RNA and for genome editing by the engineered V-type nuclease or B-GEn polypeptide as guided by the guide RNA.

In some embodiments, lipid nanoparticles (LNPs) are used to deliver engineered V-type endonucleases or B-GEn polypeptide systems (e.g., LNPs comprising one or more nucleic acids encoding engineered V-type endonucleases or B-GEn polypeptides and guide RNAs or nucleic acids encoding guide RNAs) to cells so that their genomes can be edited. In some embodiments, the method of using one or more viruses to perform genomic DNA editing includes contacting a target cell containing the genomic DNA with the lipid nanoparticle and exposing the target cell to conditions where gene editing occurs, such as by culturing the target cell under conditions suitable for expressing the engineered V-type nuclease or B-GEn polypeptide and optionally a guide RNA and performing genomic editing by the engineered V-type nuclease or B-GEn polypeptide as guided by the guide RNA.

Exemplary methods for editing a cell genome using engineered V-type endonucleases and B-GEn polypeptides are described in Section 6.13 and Examples 78-97.

Editing of a cell's genome can be performed in vitro (e.g., in cell culture), ex vivo, or in vivo (e.g., by administering RNPs, AAVs, or LNPs to an individual for the purpose of gene therapy).

Exemplary cells comprising engineered V-type endonucleases and B-GEn polypeptides and nucleic acids (e.g., cells contacted with RNPs, AAVs, or LNPs as disclosed herein) are described in Section 6.11 and in numbered Examples 98 to 110.

Additional features, advantages and applications of the engineered V-type endonucleases and B-GEn polypeptides of the present invention are described in more detail below.

1. Cross-reference to related applications This application claims priority to U.S. Provisional Application No. 63/588,636 filed on October 6, 2023, the contents of which are incorporated herein by reference in their entirety. 2. Sequence Listing

This application contains a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML sequence listing, created on September 26, 2024, is named BRT-008TW_SL.xml and is 451,061 bytes in size. 6.1. Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used to practice or test the present invention. In the event of a conflict, the present specification, including definitions, shall prevail. In general, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions as commonly accomplished in the art or as described herein. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and examples, the words "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising", should be understood to mean including the stated integer or group of integers but not excluding any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many documents are cited herein, this citation does not constitute an admission that any of these documents constitutes part of the common general knowledge in the art.

B-GEn polypeptide: As used herein, the term "B-GEn polypeptide" refers to a polypeptide comprising a nuclease domain having an amino acid sequence related to or derived from a Brevibacillus type V endonuclease, such as B-GEn.1 (SEQ ID NO: 1), B-GEn.1.2 (SEQ ID NO: 2), or B-GEn.2 (SEQ ID NO: 3). B-GEn.1 (SEQ ID NO: 1) has a nuclease domain comprising RuvC I, RuvC II, and RuvC III subdomains corresponding to SEQ ID NOS: 8-10. B-GEn.1.2 (SEQ ID NO: 2) has a nuclease domain comprising RuvC I, RuvC II, and RuvC III subdomains corresponding to SEQ ID NOS: 11-13. B-GEn.2 (SEQ ID NO: 3) has a nuclease domain comprising RuvC I, RuvC II, and RuvC III subdomains corresponding to SEQ ID NOs: 11-13. The term "B-GEn polypeptide" encompasses a polypeptide comprising an amino acid sequence having at least 40% sequence identity with the RuvC I, RuvC II, and RuvC III domains (individually or collectively) of any of B-GEn.1, B-GEn1.2, and B-GEn.2. "B-GEn polypeptide" also encompasses variants of any of B-GEn.1 (SEQ ID NO: 1), B-GEn.1.2 (SEQ ID NO: 2), B-GEn.2 (SEQ ID NO: 3), such as variants comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and/or (2) variants comprising an amino acid sequence that differs by up to 25 amino acids from SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the B-GEn polypeptide has nuclease activity. The term "B-GEn polypeptide" encompasses engineered fusion polypeptides comprising the amino acid sequence of B-GEn.1 (SEQ ID NO: 1), B-GEn.1.2 (SEQ ID NO: 2), B-GEn.2 (SEQ ID NO: 3), or any variant thereof as described in Section 6.2, such as (1) having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nuclease domain or the entire length of any one of SEQ ID NOS: 1 to 3 and/or (2) having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nuclease domain or the entire length of any one of SEQ ID NOS: Any of 1 to 3 differs by up to 25 amino acids, up to 20 amino acids, up to 15 amino acids, up to 14 amino acids, up to 13 amino acids, up to 12 amino acids, up to 11 amino acids, up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids or up to 5 amino acids, as well as additional sequences (e.g., one or more nuclear localization and/or linker sequences as described in Section 6.3 and/or Section 6.4). The term "B-GEn polypeptide" encompasses variants in which the amino acid at the position corresponding to D504 of B-GEn.1 (SEQ ID NO: 1) or D501 of B-GEn.1.2 (SEQ ID NO: 2) or D501 of B-GEn.2 (SEQ ID NO: 3) is not aspartic acid, for example, is arginine.

Binding: As used herein, the term "binding" (e.g., with reference to the RNA binding domain of a polypeptide) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). When in a state of non-covalent interaction, the macromolecules are said to be "associated" or "interacting" or "binding" (e.g., when molecule X is said to interact with molecule Y, it means that molecule X binds to molecule Y in a non-covalent manner). All components of the binding interaction need not be sequence-specific (e.g., contacts with phosphate residues in the DNA backbone), but some portions of the binding interaction may be sequence-specific. Binding interactions are typically characterized by a binding constant ^{(Kd) of less than 10-6 M, less than 10-7 M, less than 10-8 M, less than 10-9 M, less than 10-10 M , less than 10-11 M} , less than ^10-12 M, less than 10-13 M, less than ^10-14 M, or less than ^10-15 M. "Affinity" refers to the strength of binding, with increased binding affinity being associated with lower Kd.

Cell therapy: As used herein, the term "cell therapy" refers to a therapy in which a cellular material is administered to a patient. The cellular material can be intact, living cells. For example, T cells capable of fighting cancer cells via cell-mediated immunity can be administered during immunotherapy. Cell therapy is also known as cellular therapy/cytotherapy.

Coding sequence: As used herein, the term "coding sequence" or "coding nucleic acid" refers to a sequence within a nucleic acid (RNA or DNA) molecule that encodes a protein or RNA molecule. The coding sequence may further include start and stop signals that may be operably linked to regulatory elements, including promoters and polyadenylation signals that are capable of directing expression in cells of an individual or mammal into which the nucleic acid is introduced or administered. The coding sequence may be codon optimized for expression in the cell of interest.

Complementarity: As used herein, the terms "complementarity" and "complementarity" in the context of nucleic acid molecules refer to the ability of nucleotides or nucleotide analogs of nucleic acid molecules to form Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing. "Complementarity" refers to a property shared between two nucleic acid sequences such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

Corresponding/Corresponds to: The term "corresponding to/corresponds to" as used with respect to a position in a reference sequence (e.g., the amino acid sequence of a B-GEn polypeptide of any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or the RuvC I, RuvC II, and RuvC III subdomains of the nuclease domain thereof of any one of SEQ ID NO: 8 or SEQ ID NO: 11 (RuvC I), SEQ ID NO: 9 or SEQ ID NO: 12 (RuvC II), and SEQ ID NO: 10 or SEQ ID NO: 13 (RuvC II)) refers to the sequence position in the query sequence that appears at the same position in an alignment of the reference and query sequences, such as shown in Figure 1. A sequence alignment algorithm such as, for example, Clustal Omega (ClustalW; available at www.ebi.ac.uk/Tools/msa/clustalo/) can be used to align reference and query sequences using the software default parameters as of October 1, 2023.

Electroporation: As used herein, the term "electroporation" refers to a transfection technique in which electric pulses are used to create temporary pores in the cell membrane to allow the introduction of ribonucleoproteins or nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into the cell.

Coding: The term "coding" with respect to a nucleic acid (DNA or RNA) means that the nucleic acid comprises a nucleotide sequence of amino acids for a polypeptide or nucleotides for RNA.

Engineered B-GEn polypeptides: As used herein, the term "engineered B-GEn polypeptides" refers to variant polypeptides comprising at least one mutation (e.g., amino acid insertion, deletion, or substitution) relative to the amino acid sequence of B-GEn.1 (SEQ ID NO: 1), B-GEn.1.2 (SEQ ID NO: 2), and B-GEn.2 (SEQ ID NO: 3). The engineered B-GEn polypeptides of the present invention encompass V-type nucleases comprising amino acids other than aspartic acid at positions corresponding to D504 of B-GEn.1 (SEQ ID NO: 1), B-GEn.1.2 (SEQ ID NO: 2), and B-GEn.2 (SEQ ID NO: 3). In some embodiments, the amino acid at position 501 is arginine.

Expression cassette: As used herein, the term "expression cassette" refers to a DNA coding sequence that can be operably linked to a promoter.

Guide RNA: As used herein, the term "guide RNA" refers to a ribonucleic acid having a DNA targeting sequence (also referred to as a "spacer" or "DNA targeting segment") and a protein binding sequence (also referred to as a "protein binding segment"). The DNA targeting sequence is sufficiently complementary to the target DNA (e.g., genomic DNA) sequence to hybridize with the target DNA sequence and direct sequence-specific binding of the nucleic acid targeting complex to the target DNA sequence. The DNA targeting sequence generally includes a "prototype spacer-like" sequence described herein. The protein binding sequence interacts with a site-specific modifying enzyme (e.g., a B-GEn polypeptide as described in Section 6.2 below). Site-specific cleavage of the target DNA occurs at a position determined by (i) base pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif in the target DNA, called a protospacer adjacent motif (PAM). The protein-binding segment portion of the guide RNA includes two complementary stretches of nucleotides that hybridize with each other to form a double-stranded RNA duplex (dsRNA duplex). In some embodiments, the guide RNA is a single-stranded guide RNA (sgRNA).

The guide RNA and the site-specific modifying enzyme (such as a B-GEn polypeptide) can form a ribonucleoprotein complex (e.g., bound via non-covalent interactions). The guide RNA provides target specificity to the complex by comprising a nucleotide sequence that is complementary to the sequence of the target DNA. The site-specific modifying enzyme of the complex provides endonuclease activity. In other words, the site-specific modifying enzyme is guided to the target DNA sequence (e.g., a target sequence in chromosomal nucleic acid; a target sequence in an extrachromosomal nucleic acid (e.g., an episomal nucleic acid, a microcircle, etc.); a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plastid; etc.) because it binds to the protein binding segment of the guide RNA.

Heterologous: As used herein, the term "heterologous" refers to a nucleotide or peptide that is not found in a natural nucleic acid or polypeptide, respectively. The B-GEn.1, or B-GEn.1.2, or B-GEn.2 fusion protein described herein may, in some embodiments, comprise a fusion of the RNA binding domain of a B-GEn.1, or B-GEn.1.2, or B-GEn.2 polypeptide (or a variant thereof) with a heterologous polypeptide sequence (e.g., a polypeptide sequence from a protein other than B-GEn.1 or B-GEn.2). The heterologous polypeptide may exhibit an activity (e.g., an enzymatic activity) that would also be exhibited by the B-GEn.1, or B-GEn.1.2, or B-GEn.2 fusion protein (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.). A heterologous nucleic acid can be linked to a naturally occurring nucleic acid (or a variant thereof) (e.g., by genetic engineering) to produce a fusion nucleic acid encoding a fusion polypeptide. As another example, in a fusion variant B-GEn.1, or B-GEn.1.2, or B-GEn.2 polypeptide, the variant B-GEn.1, or B-GEn.1.2, or B-GEn.2 polypeptide can be fused to a heterologous polypeptide (e.g., a polypeptide other than B-GEn.1 or B-GEn.2) that exhibits an activity that will also be exhibited by the fusion variant B-GEn.1, or B-GEn.1.2, or B-GEn.2 polypeptide. A heterologous nucleic acid can be linked to a variant B-GEn.1, or B-GEn.1.2, or B-GEn.2 polypeptide (e.g., by genetic engineering) to generate a nucleic acid encoding a fusion variant B-GEn.1, or B-GEn.1.2, or B-GEn.2 polypeptide. "Heterologous" as used herein further refers to a nucleotide or polypeptide in a cell other than its natural cell.

Host cell: As used herein, the terms "host cell" and "recombinant host cell" refer to cells that have been genetically engineered, for example, by the introduction of heterologous polypeptides or nucleic acids (such as vectors or systems of the present invention). It should be understood that such terms are not intended to refer only to a specific individual cell but also to the progeny of such a cell. In some embodiments, the host cell carries the vector of the present invention as an extrachromosomal heterologous expression vector. In some embodiments, the host cell comprises any of the engineered B-GEn polypeptides disclosed herein, for example, as introduced as an RNP complex. In other embodiments, the host cell has been genetically edited by the engineered B-GEn polypeptide of the present invention.

iPSC: As used herein, the terms "induced pluripotent stem cells" and "iPSC" refer to a type of pluripotent stem cell artificially prepared from non-pluripotent cells, such as adult cells, partially differentiated cells, or terminally differentiated cells, such as fibroblasts, cells of the hematopoietic lineage, myocytes, neurons, epidermal cells, or the like, by introducing or contacting the cells with one or more reprogramming factors. iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an embryonic stem (ES) cell-like morphology, growing as flat colonies with a large nuclear-cytoplasmic ratio, defined borders, and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known to those of ordinary skill, including but not limited to alkaline phosphatases, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, Fox03, GDF3, Cyp26al, TERT, and zfp42.

Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646 and PCT Patent Publications WO2013177133 and WO2022204567, the disclosures of each of which are incorporated herein by reference. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) to reprogram the somatic cells to become pluripotent stem cells.

Nucleic Acid: As used herein, the terms "nucleic acid" and "oligonucleotide" refer to at least two nucleotides covalently linked together. Nucleic acids may be single-stranded or double-stranded or may contain portions of double-stranded and single-stranded sequences. The nucleic acid may be DNA (both genomic and cDNA), RNA, or a hybrid, wherein the nucleic acid may contain a combination of deoxyribo- and ribo-nucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isoguanosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. The description of a single strand also defines the sequence of the complementary strands. Therefore, references herein to a single-stranded nucleic acid also encompass the complementary strands of the described single strands.

Nuclear localization signal: As used herein, the terms "nuclear localization signal" and "NLS" refer to an amino acid sequence that promotes the localization of a polypeptide to the nucleus of a eukaryotic cell.

Nuclease: As used herein, the terms "nuclease" and "endonuclease" are used interchangeably herein to refer to enzymes with endonucleolytic activity for nucleic acid cleavage, as well as nuclease-inactivated variants thereof.

Nuclease domain: As used herein, the term "nuclease domain" and "cleavage domain" or "active domain" of a nuclease refers to an amino acid sequence or domain within a nuclease that has catalytic activity for DNA cleavage. The cleavage domain may be contained in a single polypeptide chain or the cleavage activity may be due to the combination of two (or more) polypeptides. A single nuclease domain may consist of more than one separate extension of amino acids within a given polypeptide. In some embodiments, the boundaries of the nuclease domain of a B-GEn polypeptide are determined by aligning the B-GEn polypeptide with BthCas12b (Wu et al., 2017, Cell Research 27:705-708) and identifying amino acids aligned with the BthCas12b RuvC nuclease domain comprising RuvC I, RuvC II, and RuvC III subdomains. The RuvC I domain of B-GEn.1 is as described in SEQ ID NO: 8, and the RuvC I domain of B-GEn.1.2 and B-GEn.2 is as described in SEQ ID NO: 11. The RuvC II domain of B-GEn.1 is as described in SEQ ID NO: 9, and the RuvC II domain of B-GEn.1.2 and B-GEn.2 is as described in SEQ ID NO: 12. The RuvC III domain of B-GEn.1 is as described in SEQ ID NO: 10, and the RuvC III domain of B-GEn.1.2 and B-GEn.2 is as described in SEQ ID NO: 13.

Nucleofection: As used herein, the term "nucleofection" refers to an electroporation-based transfection method that uses a combination of electrical parameters and cell-type specific reagents to transfer nucleic acids (such as DNA or RNA) and RNPs directly to the nucleus of a target cell.

Operably linked: As used herein, the term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence can be operably linked to a coding sequence if it stimulates or regulates transcription of the coding sequence in an appropriate host cell or other expression system.

Percent sequence identity (%): As used herein, the terms "percent sequence identity", "% sequence identity" and the like with respect to two amino acid sequences refer to the percent sequence identity as determined using the BLASTP algorithm (Tatusova and Madden, 1999, FEMS Microbiol. Lett. 174:247-250), which can be obtained from the National Center for Biotechnology Information (NCBI) website (www.ncbi.nlm.nih.gov) using the following settings: matrix = Blosum62; gap = 11; extension gap = 1; penalty gap x_down = 50; expected value = 10; word length = 3; default value. The BLAST algorithm operates in two steps by first aligning a reference sequence (e.g., a B-GEn polypeptide of any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a nuclease domain of any one of SEQ ID NO: 8, SEQ ID NO: 11 (RuvC I), SEQ ID NO: 9, SEQ ID NO: 12 (RuvC II), and SEQ ID NO: 10, or SEQ ID NO: 13 (RuvC II)) and the query sequence based thereon and then determining the % sequence identity within the overlap between the two aligned sequences. In addition to % sequence identity, BLASTP also determines % sequence similarity based on these settings. To characterize the identity, the target sequences are aligned so that the highest homology (match) is obtained.

Polypeptides, peptides and proteins: As used herein, the terms "polypeptide", "peptide" and "protein" refer to polymers of amino acids of any length. The polymer may be a linear or branched chain in various embodiments, it may contain modified amino acids, and it may be interrupted by non-amino acids.

Pluripotent: As used herein, the term "pluripotent" or "pluripotency" refers to the ability of a cell to self-renew and differentiate into cells of any of the three germ layers: endoderm, mesoderm, or ectoderm. "Pluripotent stem cells" or "PSCs" include, for example, embryonic stem cells derived from the inner cell mass of a blastocyst or by somatic cell nuclear transfer, and iPSCs derived from non-pluripotent cells.

Promoter: As used herein, the term "promoter" refers to a nucleotide sequence that is recognized by the synthetic machinery of a cell (or introduced into the synthetic machinery) required to initiate specific transcription of a nucleic acid sequence. A promoter can be a constitutively active promoter (e.g., a promoter that is constitutively in an active "on" state), it can be an induced promoter (e.g., a promoter whose state of activity/"on" or inactivity/"off" is controlled by an external stimulus, such as in the presence of a specific temperature, compound, or protein), it can be a spatially restricted promoter (e.g., a transcriptional control element, an enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), and it can be a temporally restricted promoter (e.g., the promoter is in the "on" state or the "off" state during a specific stage of embryonic development or during a specific stage of a biological process (e.g., the hair follicle cycle in mice)).

Protospacer adjacent motif: As used herein, the term "protospacer adjacent motif" or "PAM" refers to a DNA sequence downstream (e.g., immediately downstream) of a target sequence on a non-target strand that is recognized by a Cas protein. The PAM sequence is located 3' to the target sequence on the non-target strand.

Recombinant: As used herein, the term "recombinant" with respect to nucleic acids, polypeptides, or cells refers to nucleic acids (DNA or RNA), polypeptides, or cells that are the product of genetic engineering (directly or indirectly) (e.g., the progeny or replication of a nucleic acid, polypeptide, or cell produced by genetic engineering methods). For example, a recombinant vector can be the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and/or ligation steps, resulting in a construct having structural coding or non-coding sequences that are distinguishable from endogenous nucleic acids found in natural systems. A DNA sequence encoding a polypeptide can be assembled from a cDNA fragment or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid capable of being expressed from a recombinant transcription unit contained in a cell or contained in a cell-free transcription and translation system. Genomic DNA comprising the sequence of interest can also be used to form a recombinant gene or transcription unit. Sequences of non-translated DNA may be present 5' or 3' to the open reading frame, wherein such sequences do not interfere with manipulation or expression of the coding region and may in fact be used to regulate the production of a desired product by various mechanisms (see "DNA regulatory sequences" below). Additionally or alternatively, DNA sequences encoding RNA (e.g., guide RNA) that are not translated may also be considered recombinant. Thus, for example, the term "recombinant" nucleic acid refers to a non-naturally occurring nucleic acid, for example, one that is made by the artificial combination of two otherwise separate segments of sequence through human intervention. Such artificial combinations are often achieved by means of chemical synthesis or by artificial manipulation of separate nucleic acid segments, for example, by genetic engineering techniques. This is generally done by replacing codons with codons encoding the same amino acid, conservative amino acids, or non-conservative amino acids. Additionally or alternatively, nucleic acid segments of the desired functions are joined together to produce the desired functional combination. Such artificial combinations are often achieved by chemical synthesis means or by artificial manipulation of isolated nucleic acid segments, for example by genetic engineering techniques. When a recombinant nucleic acid encodes a polypeptide, the sequence of the encoded polypeptide may be naturally occurring ("wild type") or may be a variant (e.g., a mutant) of the naturally occurring sequence. Therefore, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence is not naturally occurring. On the contrary, a "recombinant" polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide may be naturally occurring ("wild type") or non-naturally occurring (e.g., a variant, mutant, etc.). Therefore, a "recombinant" polypeptide is the result of human intervention but may be a naturally occurring amino acid sequence. The term "non-naturally occurring" includes molecules that are significantly different from their naturally occurring counterparts, including chemically modified or mutated molecules.

Regulatory sequence: As used herein, the term "regulatory sequence" refers to a nucleic acid sequence required for the expression of an operably linked sequence of interest (e.g., a guide RNA or an engineered B-GEn polypeptide sequence). In some cases, the regulatory sequence may be a promoter sequence, and in other cases, the regulatory sequence may include promoter and enhancer sequences and/or other regulatory elements required for the expression of pol. The regulatory sequence may, for example, be a sequence that drives the expression of an operably linked sequence constitutively or in a tissue-specific manner.

Ribonucleoprotein (RNP) complex, ribonucleoprotein (RNP) particle: As used herein, the terms "ribonucleoprotein complex" and "ribonucleoprotein particle" refer to a complex or particle comprising a nucleoprotein and a ribonucleic acid. As provided herein, "nucleoprotein" refers to a protein capable of binding to a nucleic acid (e.g., RNA, DNA). In the case where a nucleoprotein binds to a ribonucleic acid, it is referred to as a "ribonucleoprotein". The interaction between the ribonucleoprotein and the ribonucleic acid can be direct, such as by covalent bonding, or indirect, such as by non-covalent bonding (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interactions, and the like). In an embodiment, the ribonucleoprotein includes an RNA binding motif that is non-covalently bound to the ribonucleic acid. For example, a positively charged aromatic amino acid residue (e.g., a lysine residue) in the RNA binding motif can form an electrostatic interaction with the negative nucleic acid phosphate backbone of the RNA, thereby forming a ribonucleoprotein complex. In some embodiments, any of the engineered B-GEn polypeptides disclosed herein is in an RNP with a guide RNA.

Spacer: As used herein, the term "spacer" refers to a region of a gRNA molecule that is partially or completely complementary to a target sequence found in the + or - strand of genomic DNA. When complexed with a Cas protein, the gRNA guides the Cas protein to the target sequence in the genomic DNA. The length of a spacer is typically 15 to 30 nucleotides (e.g., 20 to 25 nucleotides). The nucleotide sequence of a spacer may be (but not necessarily) completely complementary to the target sequence. For example, in some embodiments, a spacer may contain one or more mismatches with a target sequence, for example, the spacer may comprise one, two, or three mismatches with the target sequence.

Stem-ring structure: As used herein, the term "stem-ring structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides that are known or predicted to form a double strand (stem portion) that is connected on one side by a region of predominantly single stranded nucleotides (ring portion). The terms "hairpin" and "fold-back" structures are also used herein to refer to stem-ring structures. Such structures are well known in the art and these terms are used consistently with their known meanings in the art. As is known in the art, a stem-ring structure does not require exact base pairing. Thus, the stem may include one or more base mismatches. Alternatively, the base pairing may be exact, e.g., not including any mismatches.

Target cell: As used herein, the term "target cell" refers to a cell into which a nuclease (e.g., the B-GEn system of the present invention) is introduced, such as a cell that contains target DNA in its genome. It should be understood that this term is not intended to refer only to a specific individual cell but also to the progeny of such a cell. Because gene editing can be performed in the cell due to the nuclease system, this progeny does not need to be the same as the parent cell into which the system was originally introduced but includes the gene-edited counterpart of the cell. This gene-edited progeny is still included in the scope of the term "target cell" as used herein.

Target DNA: As used herein, the term "target DNA" refers to a polydeoxyribonucleotide comprising a "target site" or "target sequence". The terms "target site", "target sequence", "target protospacer DNA" or "protospacer-like sequence" are used interchangeably herein to refer to a nucleic acid sequence in the target DNA to which the DNA targeting segment (also referred to as a "spacer") of a guide RNA will bind (provided that conditions sufficient for binding exist). For example, the target site (or target sequence) 5' GAGCATATC-3' in the target DNA is targeted by (or bound by, or hybridized to, or complemented to) the RNA sequence 5'-GAUAUGCUC-3'. Suitable DNA/RNA binding conditions include physiological conditions that normally exist in cells. Other suitable DNA/RNA binding conditions (e.g., conditions in a cell-free system) are known in the art; see, e.g., Sambrook, J. and Russell, W., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press. The strand of the target DNA that complements the guide RNA and hybridizes with the guide RNA is called a "complementary strand" and the strand of the target DNA that complements the "complementary strand" (and therefore does not complement the guide RNA) is called a "non-complementary strand." In some embodiments, the target DNA is genomic DNA.

Transfection: As used herein, the term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, for example, into the nucleus of a target cell or a producer cell. In the context of the present invention, the term "transfection" encompasses any method known to the skilled artisan for introducing nucleic acid molecules into cells, such as into eukaryotic cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection (e.g., based on cationic lipids and/or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers (such as DEAE-polydextrose or polyethyleneimine). In some embodiments, the nucleic acid molecules are associated or complexed with a polypeptide, for example in the form of a ribonucleoprotein.

Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plastid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be incorporated. Another type of vector is a viral vector, in which additional DNA segments can be tethered to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after introduction into the host cell, and thereby replicate along with the host genome. In addition, certain vectors are capable of directing the expression of nucleotide sequences to which they are operably linked. Such vectors are referred to herein as "expression vectors." In some embodiments, the vector is a viral vector, such as an adenoviral vector or an adeno-associated virus (AAV) vector. 6.2. Engineered V-type endonucleases and B-GEn polypeptides

The present invention provides engineered V-type endonucleases, such as engineered Bacillusales V-type endonucleases. The engineered V-type endonucleases of the present invention generally comprise an amino acid other than aspartic acid at a position corresponding to D504 of SEQ ID NO: 1 (B-GEn. 1) or D501 of SEQ ID NO: 2 (B-GEn. 1.2) or SEQ ID NO: 3 (B-GEn. 2).

In certain aspects, the present invention also provides engineered V _{- type endonucleases} containing an OBD ( _e.g. , OBD-II) comprising a target interaction sequence motif _{GX1X2X3X4NX5X6X7DX8} (SEQ ID NO: 204), _wherein each _{of X1} to _X8 is _any amino acid. In some embodiments, (a) _X1 is selected from D, E, S, P, K, and R; (b) _X2 is independently selected from V, I, and A; (c) _X3 is independently selected from Y and F; (d) _X4 is independently selected from L and F; (e) _X5 is independently selected from I, L, F, V, and M; (f) _X6 is independently selected from S, V, T, and A; (g) _X7 is independently selected from V, L, and I; and (h) _X8 is independently selected from V, F, L, and I. In exemplary embodiments, the target interaction sequence motif is any one of SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 3.

In certain aspects, the present invention also provides an engineered V-type endonuclease, for example, an engineered V-type endonuclease comprising an OBD comprising a target interaction sequence motif as described above and/or an amino acid other than aspartic acid at a position corresponding to D504 of a V-type endonuclease of SEQ ID NO: 1 (B-GEn.1) and D501 of a V-type endonuclease of SEQ ID NO: 2 (B-GEn.1.2) and SEQ ID NO: 3 (B-GEn.2). In some embodiments, the amino acid at the position corresponding to D504 or D501 is arginine.

The engineered V-type endonucleases of the present invention generally comprise an amino acid sequence having at least 50% identity to the amino acid sequence of a V-type endonuclease of the Bacillusales, for example, any amino acid sequence having at least 50%, at least 60%, at least 70% or at least 80% identity to the amino acid sequence of any one of SEQ ID NOS _{: 1} , ₂ , ₃ , and _{179 to 199} , and contain an OBD (e.g., OBD-II) comprising the target interaction sequence motif _{GX1X2X3X4NX5X6X7DX8} (SEQ ID NO: 204), wherein each of _X1 to _X8 is any amino acid. In some embodiments, (a) _X1 is selected from D, E, S, P, K, and R; (b) _X2 is independently selected from V, I, and A; (c) _X3 is independently selected from Y and F; (d) _X4 is independently selected from L and F; (e) _X5 is independently selected from I, L, F, V, and M; (f) _X6 is independently selected from S, V, T, and A; (g) _X7 is independently selected from V, L, and I; and (h) _X8 is independently selected from V, F, L, and I. In exemplary embodiments, the target interaction sequence motif is any one of SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203.

In some aspects, the V-type endonucleases through through engineering approaches are B-GEn polypeptides through through engineering approaches. In some embodiments, the B-GEn polypeptides through through engineering approaches comprise an amino acid sequence having at least 50% identity to the entire length of B-GEn.1 (SEQ ID NO: 1), B-GEn1.2 (SEQ ID NO: 2) or B-GEn.2 (SEQ ID NO: 3) and/or differing by up to 25 amino acids from any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The amino acid sequence preferably comprises: (a) a target interaction sequence motif of any one of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203; and SEQ ID NO: 204; (b) a RuvC I domain comprising an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11; (c) a RuvC II domain comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12; (d) a RuvC III domain comprising an amino acid sequence having at least 80%, at least 85% or at least 90% sequence identity with the RuvC III domain of SEQ ID NO: 10 or SEQ ID NO: 13. III domain; or any combination of two, three, or all four of (a), (b), (c), and (d).

In some embodiments, the engineered B-GEn polypeptides of the present invention comprise an amino acid sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the entire length of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the engineered B-GEn polypeptides comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the entire length of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3. The sequence preferably comprises: (a) a target interaction sequence motif of any one of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203; and SEQ ID NO: 204; (b) a RuvC I domain comprising an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11; (c) a RuvC II domain comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12; (d) a RuvC III domain comprising an amino acid sequence having at least 80%, at least 85% or at least 90% sequence identity with the RuvC III domain of SEQ ID NO: 10 or SEQ ID NO: 13. III domain; or any combination of two, three, or all four of (a), (b), (c), and (d).

In some aspects, the engineered B-GEn polypeptides of the invention comprise (a) a target interaction sequence motif of any one of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203; and SEQ ID NO: 204 and (b) RuvC I, RuvC II, and RuvC III amino acid sequences that differ from the corresponding sequences of B-GEn.1, B-GEn.1.2, or B-GEn.2 by a total of up to 25 amino acids across three nuclease domains (SEQ ID NOS: 8 to 10 for B-GEn.1 and SEQ ID NOS: 11 to 13 for B-GEn.1.2 and B-GEn.2), optionally wherein there is no more than 3 amino acid difference in the RuvC III domain. In some embodiments, the engineered B-GEn polypeptide comprises an amino acid sequence having at least 70%, at least 80%, or at least 90% sequence identity overall to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In yet a further aspect, the engineered B-GEn polypeptide of the present invention may comprise an amino acid sequence that differs from the entire sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 by up to 25 amino acids, including the amino acid substitution D504R with respect to SEQ ID NO: 1 or D501R with respect to SEQ ID NO: 2 or SEQ NO: 3. In some embodiments, the engineered B-GEn polypeptide comprises an amino acid sequence that differs from any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 by up to 25 amino acids, up to 20 amino acids, up to 15 amino acids, up to 14 amino acids, up to 13 amino acids, up to 11 amino acids, up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, in each case including the amino acid substitution D504R with respect to SEQ ID NO: 1 or D501R with respect to SEQ ID NO: 2 or SEQ NO: 3.

The present invention provides polypeptides, such as polypeptides having one or more features as described in numbered Examples 1 to 26, which have at least 80% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or nucleic acids comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 80% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

Some embodiments according to the present invention are polypeptides, such as polypeptides having one or more of the features as described in numbered embodiments 1 to 26, which have at least 85% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or nucleic acids comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

Other embodiments according to the present invention are polypeptides, such as polypeptides having one or more of the features as specified in numbered embodiments 1 to 26, which have at least 90% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or nucleic acids comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 90% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

A further embodiment according to the present invention is a polypeptide, for example a polypeptide having one or more of the features as specified in numbered embodiments 1 to 26, which has at least 95% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case comprises the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case comprises the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

A further embodiment according to the present invention is a polypeptide, for example a polypeptide having one or more of the features as specified in numbered embodiments 1 to 26, which has at least 96% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case comprises the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 96% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case comprises the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

Another embodiment according to the present invention is a polypeptide, for example a polypeptide having one or more of the features as specified in numbered embodiments 1 to 26, which has at least 97% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case comprises the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 97% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case comprises the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

Other additional embodiments according to the present invention are polypeptides, such as polypeptides having one or more of the features as specified in numbered embodiments 1 to 26, which have at least 98% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or nucleic acids comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 98% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

Still other embodiments according to the present invention are polypeptides, such as polypeptides having one or more of the features as described in numbered embodiments 1 to 26, which have at least 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or nucleic acids comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

Still other embodiments according to the present invention are polypeptides, such as polypeptides having one or more of the features as described in numbered embodiments 1 to 26, which have at least 99.5% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3, or nucleic acids comprising a nucleotide sequence encoding a polypeptide comprising a nuclease sequence having at least 99.5% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and in each case include the amino acid arginine at a position corresponding to D504 of SEQ ID NO: 1 or at a position corresponding to D501 of SEQ ID NO: 2 or SEQ NO: 3.

Exemplary B-GEn consensus sequences B-GEn consensus I and B-GEn consensus II are provided herein as SEQ ID NO: 7 and SEQ ID NO: 205, respectively.

Exemplary engineered B-GEn sequences referred to herein as B-GEn.1 D504R (SEQ ID NO: 4), B-GEn.1.2 D501R (SEQ ID NO: 5), and B-GEn.2 D501R (SEQ ID NO: 6) are described in Table 1 below. Table 1 Name sequence SEQ ID NO B-GEn.1 D504R aa sequence 4 B-GEn.1.2 D501R aa sequence 5 B-GEn.2 D501R aa sequence 6

In some embodiments, the engineered B-GEn polypeptide comprises an amino acid sequence identical to the exemplary engineered B-GEn polypeptide of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.

In some embodiments, the polypeptides further comprise a nuclear localization signal, such as described in Section 6.3 and/or a linker sequence, such as described in Section 6.4. 6.3. Nuclear localization signal

The V-type endonuclease and B-GEn polypeptide of the present invention through through engineering approaches may further include one or more nuclear localization signals (NLS). In some embodiments, the V-type endonuclease and B-GEn polypeptide of the present invention through through engineering approaches are included in one or more NLS at its N-terminus. In some embodiments, the V-type endonuclease and B-GEn polypeptide of the present invention through through engineering approaches are included in one or more NLS at its C-terminus. In some embodiments, the V-type endonuclease and B-C-GEn polypeptide of the present invention through through engineering approaches are included in one or more NLS at its N-terminus and C-terminus. These NLS can be separated from these endonuclease sequences and separated from each other by a linker sequence.

Thus, the present invention provides engineered V-type endonucleases and B-GEn polypeptides comprising: (i) an engineered V-type endonuclease or B-GEn polypeptide sequence as described in Section 6.2; (ii) one or more NLS sequences as described herein (e.g., in Section 6.3); and (iii) one or more optional linker sequences, e.g., as described in Section 6.4.

In certain aspects, the engineered V-type endonuclease or B-GEn polypeptide comprises a nuclease sequence and a first NLS sequence at the C-terminus of the nuclease sequence. The engineered V-type endonuclease or B-GEn polypeptide comprising a nuclease sequence and a first NLS sequence may further comprise a first linker sequence between the nuclease sequence and the first NLS sequence.

In certain aspects, the engineered V-type endonuclease or B-GEn polypeptide comprises more than one NLS sequence (eg, more than one NLS sequence located at the C-terminus of the nuclease sequence).

In some embodiments, the engineered V-type endonuclease or B-GEn polypeptide comprises a second NLS sequence located at the C-terminus of the first NLS sequence. The engineered V-type endonuclease or B-GEn polypeptide comprising the second NLS sequence may further comprise a linker sequence between the first NLS sequence and the second NLS sequence.

In a further embodiment, the engineered V-type nuclease or B-GEn polypeptide comprises a third NLS sequence located at the C-terminus of the second NLS sequence. The engineered V-type nuclease or B-GEn polypeptide sequence comprising the third NLS sequence may further comprise a linker sequence between the second NLS sequence and the third NLS sequence.

In another embodiment, the engineered V-type nuclease or B-GEn polypeptide comprises a fourth NLS sequence located at the C-terminus of the third NLS sequence. The engineered V-type nuclease or B-GEn polypeptide comprising the fourth NLS sequence may further comprise a linker sequence between the third NLS sequence and the fourth NLS sequence.

In some embodiments, in addition to the one or more NLS sequences at the C-terminus of the nuclease sequence, the engineered V-type nuclease or B-GEn polypeptide also comprises an N-terminal NLS sequence. Therefore, in some aspects, the engineered V-type nuclease or B-GEn polypeptide comprises an NLS sequence at the N-terminus of the nuclease sequence. The engineered V-type nuclease or B-GEn polypeptide comprising an NLS sequence at the N-terminus of the nuclease sequence may further comprise a linker sequence between the NLS sequence and the nuclease sequence. In some embodiments, the engineered V-type nuclease or B-GEn polypeptide comprises more than one N-terminal NLS sequence (e.g., more than one NLS sequence that can be connected by one or more linkers at the N-terminus of the nuclease sequence).

Non-limiting examples of nuclear localization signals are listed in Table 2. Table 2 NLS sequence NLS SEQ ID NO PKKKRKV Wild-type SV40 large T protein 14 KRPAATKKAGQAKKKK Nucleoplasm 15 PAAKRVKLD c-myc 16 MSRRRKANPTKLSENAKKLAKEVEN EGL-13 17 KLKIKRPVK TUS 18 (KR)-X(0,2)-(KR)-(KR)-x(3,10)-(RHK)-X(1,5)-PY Alkaline PY-NLS 19 PKKKRMV SV40 large T protein, variant 1 20 PKKKRKWEDP SV40 large T protein, variant 2 twenty one CGYGPKKKRKVGG SV40 large T protein, variant 3 twenty two CGYGPKKKRKV SV40 large T protein, variant 4 twenty three CYDDEATADSQHSTPPKKKRKWEDPKDFESELLS SV40 large T protein long NLS twenty four CGGPKKKRKWG SV40 large T protein, variant 5 25 PKKKIKW SV40 large T protein, variant 6 26 KRTADGSEFESPKKKRKV SV40 large T protein, variant 7 27 TKKAGQAKKK Nucleoplasmic min-NLS 28 TKKAGQAKKKKLD Nucleocytoplasmic NLS variant 1 29 CGQAKKKKLD Nucleocytoplasmic NLS variant 2 30 RQRRNELKRSP c-myc NLS2 31 PKKARED Polyoma large T protein 32 CGYGWSRKRPRPG Polyomavirus large T protein 33 APTKRKGS SV40 VP1 capsid polypeptide 34 APKRKSGVSKC Polyomavirus major capsid protein VP1 (11 N-terminal aa) 35 PNKKKR SV40 VP2 capsid protein (39 kD) 36 EEDGPQKKKRRL Polyomavirus capsid protein VP2 37 GKKRSKA Yeast Histone H2B 38 KRPRP Adenovirus E1a 39 CGGLSSKRPRP Adenovirus type 2/5 E1a 40 LVRKKRKTE3SP Xenopus N1, NLS1 41 LKDKDAKKSKQE Xenopus N1, NLS2 42 GNKAKRQRST V-Rel 43 PFLDRLRRDQK NS1 protein of influenza A virus 44 SVTKKRKLE Human laminin A 45 SASKRRRLE Xenopus laminin A 46 ACIDKRVKLD Human c-myc 47 SALIKKKKKMAP Mouse c-abl 48 PPKKRMRRRIE Adenovirus 5 DBP 49 YRKCLQAGMNLEARKTKKKIKGIQQATA Rat glucocorticoid receptor 50 CGYGARKTKKKIK Human glucocorticoid receptor 51 RKCLQAGMNLEARKTKK Human glucocorticoid receptor NLS variants 52 RKFKKFNK Rabbit progesterone receptor 53 CGYGIRKDRRGGR Human estrogen receptor 54 CGYGARKLKKLGN Human androgen receptor 55 GKRKNKPK Chicken Ets1 nuclear NLS 56 PLLKKIKQ c-myb 57 PPQKKIKS N-myc 58 PQPKKKP p53 59 PQPKKKPL p53 NLS variants 60 SKRWAKRKL c-erb-A 61 MTGSKTRKHRGSGA Yeast ribosomal protein L29 NLS 62 MTGSKHRKHPGSGA Yeast ribosomal protein L29 NLS, variant 1 63 R Yeast ribosomal protein L29 NLS, variant 2 64 KRW Yeast ribosomal protein L29 NLS, variant 3 65 KYRKHP Yeast ribosomal protein L29 NLS, variant 4 66 KHRR Yeast ribosomal protein L29 NLS, variant 5 67 KHKKHP Yeast ribosomal protein L29 NLS, variant 6 68 RHLK Yeast ribosomal protein L29 NLS, variant 7 69 KHRKYP Yeast ribosomal protein L29 NLS, variant 8 70 QUR Yeast ribosomal protein L29 NLS, variant 9 71 LVRKKRKTE3SP Xenopus N1 NLS1 72 LKDKDAKKSKQE Xenopus N1 NLS2 73 ASKSRKRKL VirusJun 74 GGLCSARLHRHALLAT Human T-cell leukemia virus Tax transactivator protein 75 DTREKKKFLKRRLLRLDE Mouse nuclear MX1 protein (72 kD) 76 REKKKFLKRR Mouse nuclear MX1 protein NLS variant 77 CGYGDRNKKKKE Human retinoic acid receptor 78 RKRQRALMLRQAR Human XPAC 79 EYLSRKGKLEL VirD2 endonuclease for T-DNA ligation 80 KKSKKKRC Putative core NLS of yeast TRM1 206 QPQRYGGGRGRRW Human foamy retrovirus Gag protein 81 NKKKRKLSRGSSQKTKGTSASAKARH KRRNRSSRS SV40 Vp3 structural protein 82 RVTIRTWRWRRPPKGKHRK Simian sarcoma virus v-sis gene product 83 KRKIEEPEPEPKKAK Putative double NLS of Xenopus laevis protein factor Xnf7 84 KKYENVVIKRSPRKRGRPRKD Yeast SWI5 gene product 85 GRKRAFHGDDPFGEGPPDKKGD Herpes simplex virus ICP8 protein 86 KRPREDDDGEPSERKRARDDR VirD2 endonuclease double NLS 87 RMRIZFKNKGKDTAELRRRRVEVSVEL RKAKKDEQILKRRNV IBB domain from importin-α 88 NQSSNFGPMKGGNFGGRSSGPYGGG GQYFAKPRNQGGY hRNPAl M9 NLS 89 KRKGDEVDGVDEVAKKKSKK Human poly (ADP-ribose) polymerase 90 RKLKKKIKKL Hepatitis virus delta antigen 91 PKKKRK Influenza virus NLS 92 SALIKKKKKMAP Mouse c-abl IV 93 VSRKRPRP Myoma T protein NLS1 94 PPKKARED Myoma T protein NLS2 95 GPAAKRVKLD myc proto-oncogene protein [Homo sapiens] 96 KKRRIK Heat shock factor protein HSF8 [Tomato] 97 PKKKRKVEDPKKKRKVD 2x SV40, LrgT 98 PKKKRKVDPKKKRKVDPKKKRKV 3x SV40, LrgT 99 KKGKKKGK Single NLS Common Collection 1 (Dissertation Tatyana Goldberg 2016) 100 PKRRRGVVL Single NLS Common Collection 2 (Dissertation Tatyana Goldberg 2016) 101 EQLFKRRNV Single NLS Common Collection 3 (Dissertation Tatyana Goldberg 2016) 102 KRRRR Single NLS Common Collection 4 (Dissertation Tatyana Goldberg 2016) 103 KKRR Single NLS Common Collection 5 (Dissertation Tatyana Goldberg 2016) 104 EGAPPAKRPR Single NLS Common Collection 6 (Dissertation Tatyana Goldberg 2016) 105 MLRRRRRKRAR Single NLS Common Collection 7 (Dissertation Tatyana Goldberg 2016) 106 RRKRR Single NLS Common Collection 8 (Dissertation Tatyana Goldberg 2016) 107 RKR Single NLS Common Collection 9 (Dissertation Tatyana Goldberg 2016) 108 FKAVLEDILGEL Single-linked NLS minor cluster (Dissertation Tatyana Goldberg 2016) 109 KNRRL NLSdb, protein source P10152 110 R NLSdb, protein source Q09353 111 RRKKRR NLSdb, protein source Q0VD86, Q58DS6, Q5R6G2, Q9ERI5, Q6AYK2, Q6NYC1 112 RRKRSR NLSdb, protein sources Q99PU7, D3ZHS6, Q92560, A2VDM8 113 KRKP NLSdb, protein source Q14781, P30658 114 KKRKLE NLSdb, protein sources P02545, P48678, P48679, Q3ZD69 115 PKKKSRK NLSdb, protein source O35914, Q01954 116 PKRGRGR NLSdb, protein source Q9FYS5, Q43386 117 KEKRKKR NLSdb, protein source E5RQA1 118 KKKKRKR NLSdb, protein source Q9Z1J1, Q9HCS4, Q924A0 119 RRGDGRRR NLSdb, protein source Q80WE1, Q5R9B4, Q06787, P35922 120 LSPSLSPL NLSdb, protein source Q9Y261, P32182, P35583 121 VNFSEFSK NLSdb, protein source P07156 122 IVINILSE NLSdb, protein source Q96EB6 123 PPAKRKCIF NLSdb, protein source Q6AZ28, O75928, Q8C5D8 124 QRPGPYDRP NLSdb, SeqNLS prediction 125 KRKRGRPRK NLSdb, protein source Q8L7L5, A1L4X7, O80834, Q8LPN5 126 KIKELYRR NLSdb, protein source O88907, O75925 127 MVQLRPRASR NLSdb, SeqNLS prediction 128 KKRREKQRRR NLSdb, protein source Q5VK71 129 EGAPPAKRAR NLSdb, protein source P0C6L6, P25880, Q81835, P29833, P25882, P06934, P0C6L3, P29997, P0C6M5, P0C6M9, P29996, P0C6M1, P0C6L8, P0C6M2, P0C6L7, P25881 130 PKKGDKYDKTD NLSdb, protein source Q45FA5 131 KKKKSKDKKRK NLSdb, protein source P97376, Q14331 132 AHRAKKMSKTHA NLSdb, protein source P21827 133 KKGPSVQKRKKT NLSdb, protein source Q6ZN17 134 KGVKRKADTTTP NLSdb, protein source Q4R8Y1 135 KGVKRRADTTTP NLSdb, protein source Q91Y44, D4A7T3 136 KKPKWDDFKKKKK NLSdb, protein source Q15397, Q8BKS9, Q562C7 137 KRRRRRRREKRKR NLSdb, protein source Q96GM8 138 DVRKRVQDLEQKM NLSdb, protein source P61635, Q6DV79, Q19S50, P52631 139 KKGKDEWFSRGKK NLSdb, protein source O60716 140 KKGKDEWFSRGKKP NLSdb, protein source P30999 141 ASPEYVNLPINGNG NLSdb, SeqNLS prediction 142 YLRPVKKPKIRRKK NLSdb, protein source Q7Z7C8, Q5ZMS1, Q9EQH4, A7MAZ4 143 KRKGKLKNKGSKRKK NLSdb, protein source O15381 144 RRRGKNKVAAQNCRK NLSdb, SeqNLS prediction 145 DKAKRVSRNKSEKKRR NLSdb, protein source O15516, Q5RAK8, Q91YB2, Q91YB0, Q8QGQ6, O08785, Q9WVS9, Q6YGZ4 146 EEQLRRRKNSRLNNTG NLSdb, protein source G5EFF5 147 HKKKHPDASVNFSEFSK NLSdb, protein source P10103, Q4R844, P12682, B0CM99, A9RA84, Q6YKA4, P09429, P63159, Q08IE6, P63158, Q9YH06, B1MTB0 148 KKTGKNRKLKSKRVKTR NLSdb, protein source Q9Z301, O54943, Q8K3T2 149 KRSCRRRLAGHNERRRK NLSdb, protein sources Q38740, Q38741, Q700W2, Q9S7A9, Q6Z461, P93015, Q94JW8, Q9S758 150 KRQRRKQSNRESARRSR NLSdb, protein source Q501B2 151 RGKGGKGLGKGGAKRHRK NLSdb, SeqNLS prediction 152 RRRGFERFGPDNMGRKRK NLSdb, protein source Q63014, Q9DBR0 153 RRHQQGQGDDSSHKKERK NLSdb, protein source Q0IJ08, Q2TAE3, Q63470, Q13627, Q61214 154 KKKTGVIAPKRFVQRLKK NLSdb, protein source Q8LAM0, O24454 155 KRAMKDDSHGNSTSPKRRK NLSdb, protein source Q0E671 156 KVNFLDMSLDDIIIYKELE NLSdb, protein source Q9P127 157 KKYENVVIKRSPRKRGRPRK NLSdb, SeqNLS prediction 158 KRGNSSIGPNDLSKRKQRKK NLSdb, SeqNLS prediction 159 KRASEDTTSGSPPKKSSAGPKR NLSdb, protein source Q9BZZ5, Q5R644 160 KRIHSVSLSQSQIDPSKKVKRAK NLSdb, SeqNLS prediction 161 EVLKVIRTGKRKKKAWKRMVTKVC NLSdb, SeqNLS prediction 162 IINGRKLKLKKSRRRSSQTSNNSFTSRRS NLSdb, SeqNLS prediction 163 AHFKISGEKRPSTDPGKKAKNPKKKKKKDP NLSdb, protein source Q76IQ7 164

In some embodiments, the engineered B-GEn polypeptide comprises one or more NLS sequences located only at the N-terminus of the B-GEn.2 protein sequence. In other embodiments, the fusion protein of the present invention comprises one or more NLS sequences located only at the C-terminus of the B-GEn.2 protein sequence. An exemplary engineered B-GEn polypeptide with a C-terminal NLS is represented by SEQ ID NO: 200.

In some embodiments, the engineered B-GEn polypeptide comprises multiple identical NLS sequences. In other embodiments, the fusion protein of the present invention comprises multiple different NLS sequences.

In some embodiments, the engineered B-GEn polypeptide comprises a nucleoplasmic NLS (SEQ ID NO: 15) at the N-terminus of the B-GEn polypeptide sequence as described in Section 6.2 and a SV40 large T protein NLS (SEQ ID NO: 14) at the C-terminus of the B-GEn polypeptide sequence as described in Section 6.2.

In some embodiments, the engineered B-GEn polypeptide comprises the SV40 large T protein NLS (SEQ ID NO: 14) at the C-terminus of the B-GEn polypeptide sequence described in Section 6.2.

In some embodiments, the engineered B-GEn polypeptide comprises a nucleoplasmic NLS (SEQ ID NO: 15) at the N-terminus of the B-GEn polypeptide sequence as described in Section 6.2. 6.4. Linker sequence

The present invention provides engineered B-GEn polypeptides in the form of fusion proteins comprising a B-GEn polypeptide sequence (eg, having an amino acid sequence as described in Section 6.2) and one or more NLS sequences, optionally fused via a peptide linker.

In some embodiments, the B-GEn polypeptide sequence is linked to an NLS sequence at its N-terminus and/or C-terminus via a peptide linker. In other embodiments, the B-GEn polypeptide sequence is linked to one or more NLS sequences, such as between two NLS sequences or between an NLS sequence and a B-GEn polypeptide sequence, via a peptide linker that links a pair of individual polypeptide sequences.

In some embodiments, the B-GEn polypeptide of the present invention comprises multiple NLS sequences connected by the same linker. In other embodiments, the B-GEn polypeptide of the present invention comprises multiple NLS sequences connected by different linkers.

Peptide linkers suitable for use in the B-GEn polypeptides of the present invention include those disclosed in Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369. Non-limiting examples of such linkers are reproduced in Table 3 below. Table 3 Connector sequence Connection subtype SEQ ID NO (GGGGS) ₃ Flexibility 165 GGG(EAAAK) ₃ mix 166 A(EAAAK) ₄ ALE(EAAAK) ₄ A Rigidity 167 GGGGSLVPRGSGGGGS Flexibility 168 GAAPAAAPAKQEAAAAPAPAAKAAEAPAAAPAAKA Rich in proline, rigid, interdomain 169 HRQPRGWE Cuttable 170 6.5. B-GEn V-type CRISPR-Cas system

The present invention provides engineered V-type endonucleases and B-GEn polypeptide systems incorporating the engineered V-type endonucleases or B-GEn polypeptides of the present invention or nucleic acids encoding the same.

In some embodiments, the engineered V-type nuclease or B-GEn polypeptide system comprises the following components: (a)  An engineered V-type nuclease or B-GEn polypeptide, such as described in Section 6.2, or a nucleic acid encoding such an engineered B-GEn polypeptide, such as described in Section 6.8, (b) A heterologous guide RNA (gRNA), such as described in Section 6.6, or a nucleic acid that allows the in situ generation of such a gRNA (such as a vector as described in Section 6.9), wherein the gRNA comprises: i. An engineered DNA targeting segment consisting of RNA and capable of hybridizing to a target sequence in a nucleic acid locus, ii. A tracr pairing sequence consisting of RNA, and iii. A tracr RNA sequence consisting of RNA, The tracr mate sequence is hybridized to the tracr sequence, and (i), (ii) and (iii) are arranged in a 5' to 3' orientation. The gRNA may be a single guide RNA (sgRNA). In the sgRNA, the tracr mate sequence and the tracr sequence are generally connected by a suitable loop sequence to form a stem-loop structure.

In certain embodiments, the engineered V-type endonuclease or B-GEn polypeptide system comprises the following components: (a)  For example, an engineered V-type endonuclease or B-GEn polypeptide as described in Section 6.2, (b) For example, a heterologous guide RNA (gRNA) as described in Section 6.6, comprising: i. An engineered DNA targeting segment consisting of RNA and capable of hybridizing to a target sequence in a nucleic acid locus, ii. A tracr mate sequence consisting of RNA, and iii. A tracr RNA sequence consisting of RNA, wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation. The gRNA can be a single guide RNA (sgRNA). In sgRNA, the tracr mate sequence and the tracr sequence are generally connected by an appropriate loop sequence to form a stem-loop structure.

In some embodiments, such engineered V-type endonucleases or B-GEn polypeptides are delivered to target cells as compositions known as ribonucleoprotein (RNP) complexes as described in Section 6.7.

In certain embodiments, the engineered V-type endonuclease or B-GEn polypeptide system comprises the following components: (a) a nucleic acid encoding an engineered V-type endonuclease or B-GEn polypeptide, such as described in Section 6.8, or (b) a nucleic acid (such as a vector as described in Section 6.9) that allows the production of a heterologous guide RNA (gRNA), such as described in Section 6.6, wherein the gRNA comprises: i. an engineered DNA targeting segment consisting of RNA and capable of hybridizing to a target sequence in a nucleic acid locus, ii. a tracr mate sequence consisting of RNA, and iii. a tracr RNA sequence consisting of RNA, wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation. The gRNA may be a single guide RNA (sgRNA). In sgRNA, the tracr pairing sequence and the tracr sequence are generally connected by a suitable loop sequence to form a stem-loop structure.

Any reference to "RNA" or "guide RNA" encompasses RNA molecules containing non-natural as well as natural nucleobases, such as one or more of the nucleic acid modifications described in Section 6.8.3.

In some embodiments, the nucleic acid encoding the engineered V-type endonuclease or B-GEn polypeptide and/or the sgRNAs contains a suitable promoter for expression in cells or in vitro environments.

In some embodiments, the nucleic acid encoding the engineered V-type endonuclease or B-GEn polypeptide and/or the sgRNAs is in the form of a viral vector, for example as described in Section 6.9.3. 6.6. Guide RNA (gRNA) and single guide RNA (sgRNA)

The systems, compositions and methods described herein in some embodiments use a genome targeting nucleic acid that can direct the activity of an engineered B-GEn polypeptide to a specific target sequence in a target nucleic acid. In some embodiments, the genome targeting nucleic acid is RNA. Genome targeting RNA is referred to herein as "guide RNA" or "gRNA". The guide RNA has at least a spacer sequence and a CRISPR repeat sequence that can be hybridized to the target nucleic acid sequence of interest (this CRISPR repeat sequence is also referred to as a "tracr pairing sequence"). In a type II system, the gRNA also has a second RNA called a tracrRNA sequence. In a type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a duplex. In a type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex binds to the site-specific polypeptide so that the guide RNA and the site-directed polypeptide form a complex. The genome-targeting nucleic acid provides target specificity to the complex by virtue of its binding to the site-specific polypeptide. Thus, the genome-targeting nucleic acid directs the activity of the site-specific polypeptide.

In some embodiments, the genome targeting nucleic acid is a bimolecular guide RNA. In some embodiments, the genome targeting nucleic acid is a single molecule guide RNA or a single guide RNA (sgRNA). Bimolecular guide RNA has two strands of RNA. The first strand has an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand has a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The single molecule guide RNA (sgRNA) in the type II system has an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension may have an element that contributes additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker connects the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA is expressed with one or more hairpins.

The single molecule guide RNA (sgRNA) in the V-type system has a minimal CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction. Or alternatively, the single molecule guide RNA (sgRNA) in the V-type system has an optional tracr extension sequence, a tracr RNA sequence, a single molecule guide linker, a minimal CRISPR repeat sequence, a spacer sequence, and an optional spacer extension sequence in the 5' to 3' direction.

Alternatively, the single-molecule guide RNA (sgRNA) in the V-type system has an optional extension sequence, a minimal CRISPR repeat sequence, a spacer sequence, and an optional spacer extension sequence in the 5' to 3' direction.

In some embodiments, the sgRNA in the V-type system comprises an optional extension sequence, an artificial nuclease binding RNA sequence and a spacer sequence, and an optional spacer extension sequence in the 5' to 3' direction.

Particularly useful sgRNAs for use with the B-GEn.2 CRISPR Cas nuclease according to the present invention, and potentially for other V-type endonuclease nucleases, are disclosed in Table 4. Table 4 sgRNA sequence SEQ ID NO B-GEn.2-sgRNA_v4 171 B-GEn.2-sgRNA_v4.2 172 B-GEn.2-sgRNA_v4.3 173 B-GEn.2-sgRNA_v4.4 174 B-GEn.2-sgRNA_v4.5 175

Exemplary genome targeting nucleic acids are described, for example, in WO2018002719. In general, a CRISPR repeat sequence includes any sequence that is sufficiently complementary to a tracr sequence to promote one or more of the following: (1) excision of a DNA targeting segment flanking the CRISPR repeat sequence in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex includes the CRISPR repeat sequence hybridized to the tracr sequence. In general, the degree of complementarity is referenced to the optimal alignment of the CRISPR repeat sequence and the tracr sequence, in terms of the length of the shorter of the two sequences. The optimal alignment can be determined by any suitable alignment algorithm and can further take into account secondary structure, such as self-complementarity within the tracr sequence or the CRISPR repeat sequence. In some embodiments, the degree of complementarity between the tracr sequence and the CRISPR repeat sequence along the 30 nucleotide length of the shorter of the two when optimally aligned is about or more than 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more. In some embodiments, the length of the tracr sequence is about or more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides. In some embodiments, the tracr sequence and the CRISPR repeat sequence are contained in a single transcript such that hybridization between the two produces a transcript having a secondary structure (e.g., a hairpin). In some embodiments, the transcript or transcribed nucleic acid sequence has at least two or more hairpins.

Suitable tracr sequences for use with B-GEn.2 or B-GEn.1 in the CRISPR Cas system are listed in Table 5. Alternatively, variants of these sequences may be used. Variants may include portions or truncated forms of these sequences and/or sequences with base modifications in one or more places in these sequences. The respective RNA sequences are disclosed in SEQ ID NOs: 176 and 177, respectively. Table 5 Name SEQ ID NO: RNA -seq tracr_B-GEn.2 176 tracr_B-GEn.1 177

The spacer of the guide RNA includes a nucleotide sequence that is complementary to a sequence in the target DNA. In other words, the spacer of the guide RNA interacts with the target DNA in a sequence-specific manner via hybridization (e.g., base pairing). Thus, the nucleotide sequence of the spacer can vary and determine the location within the target DNA where the guide RNA and target DNA will interact. The DNA targeting segment of the guide RNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within the target DNA.

In some embodiments, the spacer has a length of 10 nucleotides to 30 nucleotides. In some embodiments, the spacer has a length of 13 nucleotides to 25 nucleotides. In some embodiments, the spacer has a length of 15 nucleotides to 23 nucleotides. In some embodiments, the spacer has a length of 18 nucleotides to 22 nucleotides, such as 20 to 22 nucleotides.

In some embodiments, the percent complementarity between the DNA targeting sequence of the spacer and the protospacer of the target DNA is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) over 20 to 22 nucleotides.

In some embodiments, the protospacer is directly adjacent to a suitable PAM sequence on its 3' end or the PAM sequence is part of the DNA targeting sequence in its 3' portion.

Suitable PAM sequences are listed in Table 6, where the engineered DNA targeting segment is directly adjacent to the PAM sequence on the targeting DNA segment on its 3' end or the PAM sequence is part of the targeting DNA sequence in its 5' portion. Table 6 B-GEn PAM Sequence B-GEn.1 "DTTN", where "D" stands for "A" or "T" or "G";"ATT" is preferred B-GEn.1.2 "DTTN", where "D" stands for "A" or "T" or "G";"ATT" is preferred B-GEn.2 "DTTN", where "D" stands for "A" or "T" or "G";"ATT" is preferred

Modification of the guide RNA can be used to enhance the formation or stability of the CRISPR-Cas genome editing complex comprising the guide RNA and a Cas endonuclease (such as B-GEn.1 or B-GEn.2). Modification of the guide RNA can also or alternatively be used to enhance the initiation, stability or kinetics of the interaction between the genome editing complex and the target sequence in the genome, which can be used, for example, to enhance on-target activity. Modification of the guide RNA can also or alternatively be used to enhance specificity, for example, the relative rate of genome editing at the on-target site compared to the effect at other (off-target) sites.

Modifications may also or alternatively be used to increase the stability of the guide RNA, for example, by increasing its resistance to degradation by ribonucleases (RNases) present in the cell, thereby resulting in an increase in its half-life in the cell. Modifications that enhance the half-life of the guide RNA may be particularly useful in embodiments in which a Cas endonuclease (such as B-GEn.1, or B-GEn.1.2, or B-GEn.2) is introduced into the cell to be edited via an RNA that needs to be translated in order to produce the B-GEn.1, or B-GEn.1.2, or B-GEn.2 endonuclease, because increasing the half-life of the guide RNA introduced simultaneously with the RNA encoding the endonuclease may be used to increase the time that the guide RNAs and the encoded Cas endonuclease co-exist in the cell. 6.6.1. Other sequences

In some embodiments, the guide RNA comprises at least one additional segment at the 5' or 3' end. For example, suitable additional segments may include a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (e.g., a 3' poly (A) tail); a riboswitch sequence (e.g., to allow regulated stability and/or regulated accessibility of proteins and protein complexes); a sequence that forms a dsRNA duplex (e.g., a hairpin); a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a sequence that provides Modifications or sequences that track (e.g., directly bind to a fluorescent molecule, bind to a moiety that promotes fluorescence detection, allow fluorescence detection, etc.); modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like); modifications or sequences that provide increased, decreased, and/or controllable stability; and combinations thereof. 6.6.1.1. Stability Control Sequences

Stability control sequences affect the stability of RNA (e.g., guide RNA). A non-limiting example of a suitable stability control sequence is a transcriptional terminator segment (e.g., a transcriptional terminator sequence). The transcriptional terminator segment of the guide RNA may have a total length of 10 nucleotides to 100 nucleotides, such as 10 nucleotides (nt) to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, or 90 nt to 100 nt. For example, the transcriptional terminator segment can have a length of 15 nucleotides (nt) to 80 nt, 15 nt to 50 nt, 15 nt to 40 nt, 15 nt to 30 nt, or 15 nt to 25 nt.

In some embodiments, the transcription termination sequence is a sequence that functions in eukaryotic cells. In some embodiments, the transcription termination sequence is a sequence that functions in prokaryotic cells.

Nucleotide sequences that may be included in stability control sequences (e.g., transcriptional termination segments, or in any segment of a guide RNA to provide increased stability) include, for example, Rho-independent trp termination sites. 6.7. Ribonucleoprotein (RNP) complexes

In some embodiments, the engineered V-type endonucleases and B-GEn polypeptides are delivered as a composition called a ribonucleoprotein or RNP complex. The RNP complex is assembled by combining a Cas endonuclease (such as an engineered V-type or B-GEn endonuclease) with a ribonucleic acid (e.g., a guide RNA (gRNA)).

In some embodiments, the ribonucleoprotein complex comprises a complex of an engineered B-GEn endonuclease, such as described in Section 6.2, and a suitable ribonucleic acid. In some embodiments, the ribonucleic acid is a gRNA or sgRNA, which is further described in Section 6.6. In some embodiments, the RNP complex comprises an engineered B-GEn polypeptide and an sgRNA listed in Table 4 or another suitable sgRNA.

In some embodiments, the engineered B-GEn polypeptide and sgRNA are present in a molar ratio of about 1:1 to about 1:4. In some embodiments, the molar ratio is about 1:1 to about 1:3. In some embodiments, the molar ratio is about 1:1 to about 1:2.5. In some embodiments, the molar ratio is about 1:1 to about 1:2. In some embodiments, the molar ratio is about 1:1 to about 1:1.5. In some embodiments, the molar ratio of polypeptide to sgRNA is 1:1.

One of the most common techniques used to deliver RNPs is electroporation, which creates pores in the cell membrane, allowing the RNPs to enter the cytoplasm. In addition, electroporation can be combined with cell type-specific reagents, a technique called nucleofection, which creates pores in the nuclear membrane, allowing access to the DNA template.

In some embodiments, the engineered V-type endonuclease or B-GEn polypeptide in the RNP complex is delivered to the target cell via nuclear transfection. 6.8. Nucleic Acids

The present invention provides nucleic acids (e.g., DNA or RNA) encoding B-GEn type V CRISPR-Cas proteins (e.g., engineered B-GEn polypeptides), nucleic acids encoding gRNAs or sgRNAs of the present invention, nucleic acids encoding engineered B-GEn polypeptides and gRNAs or sgRNAs, and a plurality of nucleic acids, e.g., comprising nucleic acids encoding engineered B-GEn polypeptides and gRNAs or sgRNAs.

Nucleic acids encoding engineered B-GEn polypeptides can be codon-optimized, e.g., wherein at least one uncommon or less common codon has been replaced with a codon that is common in a host cell or target cell. For example, a codon-optimized nucleic acid can direct the synthesis of an optimized signaling mRNA, e.g., optimized for expression in a mammalian expression system. 6.8.1. B-GEn Coding Sequences

In some embodiments, the nucleic acids described herein comprise one or more modifications that can be used, for example, to enhance activity, stability or specificity, alter delivery, reduce innate immune responses in host cells, further reduce protein size, or for other enhancements, as further described herein and known in the art. In some embodiments, such modifications will produce an engineered B-GEn polypeptide whose nuclease sequence component has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to the sequence of SEQ ID NOs: 4, 5, or 6. 6.8.2. Codon Optimization

In certain embodiments, modified nucleic acids are used in the CRISPR-B-GEn.1, or B-GEn.1.2, or B-GEn.2 systems described herein, wherein guide RNAs and/or DNA or RNAs comprising nucleic acid sequences encoding engineered B-GEn polypeptides may be modified, as described below. Such modified nucleic acids may be used in CRISPR-B-GEn.1, or B-GEn.1.2, or B-GEn.2 systems to edit any one or more genomic loci. In some embodiments, such modifications in the nucleic acids of the present invention are achieved by codon optimization (e.g., codon optimization based on a specific host cell in which the encoded polypeptide is expressed). A skilled artisan will appreciate that any nucleotide sequence and/or recombinant nucleic acid of the present invention may be codon optimized for expression in any species of interest. Codon optimization is well known in the art and involves modifying nucleotide sequences for codon usage bias using species-specific codon usage tables. Such codon usage tables are generated based on sequence analysis of the most highly expressed genes of the species of interest. In a non-limiting example, when the nucleotide sequence is intended to be expressed in the nucleus, the codon usage tables are generated based on sequence analysis of highly expressed nuclear genes of the species of interest. Modifications of the nucleotide sequences are determined by comparing the species-specific codon usage table to the codons present in the natural nucleic acid sequence.

In some embodiments, the engineered B-GEn polypeptides described herein are expressed from codon-optimized nucleic acid sequences. For example, if the desired host cell or target cell is a human cell, a human codon-optimized nucleic acid sequence encoding an engineered B-GEn polypeptide comprising an amino acid sequence of B-GEn.1, or B-GEn.1.2, or B-GEn.2 (or a B-GEn.1, or B-GEn.1.2, or a B-GEn.2 variant, such as an enzyme-inactive variant) would be appropriate. As another non-limiting example, if the desired host cell or target cell is a mouse cell, a mouse codon-optimized nucleic acid sequence encoding an engineered B-GEn polypeptide comprising the amino acid sequence of B-GEn.1, or B-GEn.1.2, or B-GEn.2 (or B-GEn.1, or B-GEn.1.2, or a B-GEn.2 variant, such as an enzyme-inactive variant) would be appropriate.

Strategies and methods for codon optimization are known in the art and have been described for various systems, including, but not limited to, yeast (Outchkourov et al., Protein Expr Purif, 24(1):18-24 (2002)) and E. coli (Feng et al., Biochemistry, 39(50):15399-15409 (2000)). In some embodiments, codon optimization is performed using GeneGPS® Expression Optimization Technology (ATUM) and using the manufacturer's recommended expression optimization algorithm. In some embodiments, the nucleic acids of the invention are codon optimized to achieve increased expression in human cells. In some embodiments, the nucleic acids of the invention are codon optimized to achieve increased expression in E. coli cells. In some embodiments, nucleic acids of the invention are codon optimized to achieve increased expression in insect cells. In some embodiments, nucleic acids of the invention are codon optimized to achieve increased expression in Sf9 insect cells. In some embodiments, the performance optimization algorithm used in the codon optimization procedure is defined to avoid putative poly-A signals (e.g., AATAAA and ATTAAA) and long (greater than 4) stretches of A that can cause polymerase slippage.

As is well known in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence that is less than 100% identical (e.g., less than 70%, 71% 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to a native nucleotide sequence but still encodes a nucleotide sequence that has the same function as a polypeptide encoded by the original native nucleotide sequence. Thus, in representative embodiments of the invention, the nucleotide sequences and/or recombinant nucleic acids of the invention can be codon optimized to achieve expression in a particular species of interest.

In some embodiments, the codon-optimized nucleic acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the nucleic acids of the present invention are codon-optimized to achieve increased expression of the encoded engineered B-GEn polypeptide in target cells or host cells. In some embodiments, the nucleic acids of the present invention are codon-optimized to achieve increased expression in human cells. In general, the nucleic acids of the present invention are codon-optimized to achieve increased expression in any human cell. In some embodiments, the nucleic acids of the invention are codon optimized for increased expression in E. coli cells. In some embodiments, the nucleic acids of the invention are codon optimized for increased expression in insect cells. In general, the nucleic acids of the invention are codon optimized for increased expression in any insect cell. In some embodiments, the nucleic acids of the invention are codon optimized for increased expression in the Sf9 insect cell expression system.

Polyadenylation signals can also be selected to optimize expression in a desired host. 6.8.3. Nucleic Acid Modifications

In some embodiments, a nucleic acid (e.g., a guide RNA, a nucleic acid comprising a nucleotide sequence encoding a guide RNA; a nucleic acid encoding a site-specific modifying enzyme (e.g., an engineered B-GEn polypeptide of the present invention); etc.) includes modifications or sequences that provide additional desired characteristics (e.g., modified or regulated stability; subcellular targeting; tracking, such as a fluorescent tag; a binding site for a protein or protein complex; etc.). Non-limiting examples include: a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (e.g., a 3' poly (A) tail); a riboswitch sequence (e.g., to allow regulated stability and/or regulated accessibility of a protein and/or protein complex); a stability control sequence; a sequence that forms a dsRNA duplex (e.g., a hairpin); targeting RNA to subcellular locations (e.g., nucleus, mitochondria, chloroplasts, and and the like); modifications or sequences that provide tracking (e.g., direct binding to a fluorescent molecule, binding to a moiety that promotes fluorescence detection, a sequence that allows fluorescence detection, etc.); modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like); and combinations thereof.

In some embodiments, the guide RNA includes an additional segment at the 5' or 3' end that provides any of the above characteristics. For example, a suitable third segment may include a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (e.g., a 3' poly (A) tail); a riboswitch sequence (e.g., to allow regulated stability and/or regulated accessibility of proteins and protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (e.g., a hairpin); a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts); and the like); modifications or sequences that provide tracking (e.g., direct binding to a fluorescent molecule, binding to a moiety that promotes fluorescence detection, sequences that allow fluorescence detection, etc.); modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like); and combinations thereof.

Modifications may also or alternatively be used to reduce the likelihood or extent to which an RNA introduced into a cell elicits an innate immune response. Such responses, which have been well described in the context of RNA interference (RNAi), including small interfering RNA (siRNA), as described below and in this art, tend to be associated with a reduced half-life of the RNA and/or the induction of cytokines or other factors associated with an immune response.

The RNA encoding the engineered B-GEn polypeptide introduced into the cell may also be subjected to one or more types of modifications, including, but not limited to, modifications that enhance the stability of the RNA (such as by reducing its degradation by RNases present in the cell), modifications that enhance the translation of the resulting product (such as an endonuclease), and/or modifications that reduce the likelihood or degree of the RNA introduced into the cell eliciting an innate immune response. Combinations of modifications (such as those described above and others) may also be used. In the case of an engineered B-GEn polypeptide, for example, one or more types of modifications may be performed to guide RNA (including those listed above), and/or one or more types of modifications may be performed to RNA encoding the engineered B-GEn polypeptide (including those listed above).

By way of illustration, guide RNAs or other smaller RNAs used in the CRISPR-B-GEn system can be readily synthesized by chemical means, allowing many modifications to be readily incorporated, as described below and in this technology. Although chemical synthesis procedures continue to expand, as nucleic acid lengths increase significantly beyond a hundred or so nucleotides, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging. One method for producing chemically modified RNAs of greater length is to produce two or more molecules tethered together. Much longer RNAs (such as those encoding B-GEn.1, or B-GEn.1.2, or B-GEn.2 endonucleases) are more easily produced enzymatically. Although fewer types of modifications are generally available for use in enzymatically produced RNA, there are modifications that can be used, for example, to enhance stability, reduce the likelihood or magnitude of an innate immune response, and/or enhance other properties, as described further below and in this technology; and new types of modifications are regularly developed.

By way of illustration of various types of modifications, particularly those commonly used with smaller chemically synthesized RNAs, modifications may include one or more nucleotides modified at the 2' position of the sugar, in some embodiments 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotides. In some embodiments, RNA modifications include 2'-fluoro, 2'-amine, and 2' O-methyl modifications on the ribose of pyrimidines, basic residues, or inverted bases at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and such oligonucleotides have been shown to have higher Tm (e.g., higher target binding affinity) for a given target than 2'deoxy oligonucleotides.

Many nucleotide and nucleoside modifications have been shown to render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than native oligonucleotides; such modified oligonucleotides persist intact longer than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, such as phosphothioate, phosphotriester, methylphosphonate, short-chain alkyl or cycloalkyl sugar inter-linkages or short-chain heteroatom or heterocyclic sugar inter-linkages. Some oligonucleotides are oligonucleotides with thiophosphate backbones and those with heteroatom backbones, particularly CH2-NH-O-CH2, CH, -N(CH3)-O-CH2 (referred to as methylene (methylimino) or MMI backbones), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and ON(CH3)-CH2-CH2 backbones; amide backbones (see De Mesmaeker et al., 1995, Ace. Chem. Res., 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Patent No. 5,034,506); peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotides is replaced by a polyamide backbone, the nucleotides being directly or indirectly bound to nitrogen atoms of the polyamide backbone, see Nielsen et al., 1991, Science 254:1497). Phosphorus-containing linkages include, but are not limited to, phosphothioates, chiral phosphothioates, dithiophosphates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphamidates, including 3'-aminophosphamidates and aminoalkylphosphamidates, thiocarbonylphosphamidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2 ^{, 1} to 5' phosphodiester derivatives thereof. ¹ -linked analogs, and those with reverse polarity, wherein adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Patents Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177, 196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233 No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

Oligomers based on morpholinyl are described in Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Vol. 30, No. 3, (2001); Heasman, Dev. Biol., 243:209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacenra et al., Proc. Nat/. Acad. Sci., 97: 9591-9596 (2000); and in U.S. Patent No. 5,034,506, issued July 23, 1991. Cyclohexenyl nucleic acid oligonucleotide analogs are described in Wang et al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

The modified oligonucleotide backbones which do not include a phosphorus atom have backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those having morpholinyl linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfone and sulfonium backbones; fumaryl and thiofumaryl backbones; methylenefumaryl and thiofumaryl backbones; backbones containing olefins; sulfamate backbones; methyleneimino and methylenenitrile backbones; sulfonate and sulfonamide backbones; amide backbones; and backbones having mixed N, O, Sand and Others of the CH2 component; see U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610, No. 289; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704; No. 5,623,070; No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439, each of which is incorporated herein by reference.

It may also include one or more substituted sugar moieties at the 2' position, such as one of the following: OH, SH, SCH3, F, OCN, OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2, or O(CH2)n CH3, wherein n is 1 to 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S- or N-alkyl; O-, S- or N-alkenyl: SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacokinetic properties of oligonucleotides and other substituents with similar properties. In some embodiments, the modification includes 2'methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) (Martinet a/, Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide (particularly at the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide). Oligonucleotides may also have sugar analogs (such as cyclobutyl) substituted for the pentofuranosyl group. In some embodiments, the sugars and internucleoside linkages (e.g., the backbone) of the nucleotide units are replaced by novel groups. The base units are maintained to hybridize with the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide analog that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone (e.g., an aminoethylglycine backbone). The nucleobases are retained and are directly or indirectly bound to nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500 (1991).

The guide RNA may also include (additionally or alternatively) nucleobase (often referred to in the art as simply "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are only rarely or transiently found in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, particularly 5-methylcytosine (also known as 5-methyl-2'deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentianbiose HMC, and synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A, DNA Replication, W. H. Freeman & Co., San Francisco, pp75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). "Universal" bases known in the art, such as inosine, may also be included. 5-Me-C substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, Y. S., Crooke, S. T., and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and is an example of a base substitution.

Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine. adenine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other α-substituted adenines and guanines, 5-halogen (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Other useful nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, J.L., ed., John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, page 613, and those disclosed in Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B. et al., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and -O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2 oc (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., eds., "Antisense Research and Applications", CRC Press, Boca Raton, 1993, pp. 276-278) and is an example of a base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification. Modified nucleobases are described in U.S. Patents Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177 No. 5,525,711; No. 5,552,540; No. 5,587,469; No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 5,763,588; No. 5,830,653; No. 6,005,096; and U.S. Patent Application Publication No. 20030158403.

It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even within a single nucleoside within an oligonucleotide.

In some embodiments, the guide RNA and/or mRNA encoding the endonuclease of the present invention (such as B-GEn.1, or B-GEn.1.2, or B-GEn.2) is capped using any of the current capping methods (such as mCAP, ARCA, or enzymatic capping methods) to create a viable mRNA construct that retains biological activity and avoids self/non-self intracellular reactions. In some embodiments, the guide RNA and/or mRNA encoding the endonuclease of the present invention (such as B-GEn.1, or B-GEn.1.2, or B-GEn.2) is capped by using the CleanCap™ (TriLink) co-transcriptional capping method.

In some embodiments, the guide RNA and/or mRNA encoding the endonuclease of the present invention comprises one or more modifications selected from the group consisting of pseudouridine, N1-methylpseudouridine, and 5-methoxyuridine. In some embodiments, one or more N1-methylpseudouridines are incorporated into the guide RNA and/or mRNA encoding the endonuclease of the present invention to provide enhanced RNA stability and/or protein expression and reduced immunogenicity in animal cells, such as mammalian cells (e.g., human and mouse). In some embodiments, the N1-methylpseudouridine modifications are incorporated in combination with one or more 5-methylcytidines.

In some embodiments, the guide RNA and/or mRNA (or DNA) encoding a nuclease (such as B-GEn.1, or B-GEn.1.2, or B-GEn.2) is chemically linked to one or more moieties or binding agents that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., 1989, Proc. Nat/. Acad. Sci. USA 86: 6553-6556); cholic acid (Manoharan et al., 1994, Bioorg. Med. Chem. Let. 4: 1053-1060); thioethers, for example, hexyl-S-tritylthiol (Manoharan et al., 1992, Ann. N.Y. Acad. Sci. 660: 306-309 and Manoharan et al., 1993, Bioorg. Med. Chem. Let. 3: 2765-2770); thiocholesterol (Oberhauser et al., 1992, Nucl. Acids Res. 20: 1061-1064); 533-538); aliphatic chains, for example, dodecanediol or undecyl residues (Kabanov et al., 1990, FEBS Lett., 259: 327-330 and Svinarchuk et al., 1993, Biochimie, 75: 49-54); phospholipids, for example, di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al., 1995, Tetrahedron Lett. 36: 3651-3654 and Shea et al., 1990, Nucl. Acids Res. 18: 3777-3783); polyamines or polyethylene glycol chains (Mancharan et al., 1995, Nucleosides & Nucleotides 14:969-973); adamantane acetic acid (Manoharan et al., 1995, Tetrahedron Lett. 36:3651-3654); a palmityl moiety (Mishra et al., 1995, Biochim. Biophys. Acta 1264:229-237); or an octadecylamine or hexylamino-carbonyl-t-oxycholesterol moiety (Crooke et al., 1996, J. Pharmacol. Exp. Ther., 277:923-937). See also U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5, No. 414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5 ,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241, No. 5,391,723; No. No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928 and No. 5,688,941.

Sugars and other moieties can be used to target proteins and complexes (including nucleotides, such as cationic polysomes and liposomes) to specific sites. For example, hepatocyte-directed translocation can be mediated via the asialoglycoprotein receptor (ASGPR); see, e.g., Hu et al., 2014, Protein Pept Lett. 21(10):1025-30. Other systems known in the art and regularly developed can be used to target biomolecules and/or their complexes used in this context to specific target cells of interest.

Such targeting moieties or conjugates may include a conjugate group covalently bound to a functional group such as a primary or secondary hydroxyl group. Suitable conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacodynamic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazines, folates, phenanthridines, anthraquinones, acridines, fluoresceins, rhodamine, coumarins, and dyes. Groups that can enhance pharmacodynamic properties include groups that improve absorption, enhance resistance to degradation, and/or enhance sequence-specific hybridization with the target nucleic acid. Groups capable of enhancing pharmacokinetic properties include groups that improve the absorption, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196 filed on October 23, 1992 and U.S. Patent No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties, bile acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecandiol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-H phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties. See, e.g., U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241, No. 5,391,723; No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928 and No. 5,688,941.

Longer nucleic acids, which are less susceptible to chemical synthesis and are generally produced by enzymatic synthesis, can also be modified by various means. Such modifications may include, for example, the introduction of certain nucleotide analogs, the incorporation of specific sequences or other portions at the 5' or 3' end of the molecule, and other modifications. By way of illustration, the mRNA encoding B-GEn.1, or B-GEn.1.2, or B-GEn.2 is approximately 4 kb in length and can be synthesized by in vivo transcription. Modifications to the mRNA can be applied, for example, to increase its translation or stability (such as by increasing its resistance to degradation by cells), or to reduce the tendency of the RNA to induce an innate immune response, which is often observed in cells after the introduction of exogenous RNA (particularly longer RNA, such as that encoding B-GEn.1 or B-GEn.2).

Many such modifications have been described in the art, such as poly A tails, 5' cap analogs such as reverse cap analogs (ARCA) or m7G(5')ppp(5')G (mCAP), modified 5' or 3' untranslated regions (UTRs), use of modified bases such as pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP, or treatment with phosphatases to remove the 5' terminal phosphate. These and other modifications are known in the art, and novel modifications of RNA are regularly developed.

There are many commercial suppliers of modified RNA, including, for example, TriLink Biotech, Axolabs, Bio-Synthesis Inc., Dharmacon, and many others. As described by TriLink, for example, 5-methyl-CTP can be used to impart desired properties, such as increased nuclease stability, increased translation, or decreased interaction of innate immune receptors with transcribed RNA in vitro. 5'-methylcytidine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, as well as pseudo-UTP and 2-thio-UTP have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation, as described in the publications of Konmann et al. and Warren et al., referenced below.

It has been shown that chemically modified mRNAs delivered in vivo can be used to achieve improved therapeutic effects; see, e.g., Kormann et al., Nature Biotechnology 29, 154-157 (2011). Such modifications can be used, for example, to increase the stability of the RNA molecule and/or reduce its immunogenicity. Using chemical modifications such as pseudo-U, N6-methyl-A, 2-thio-U, and 5-methyl-C, it was found that replacing only one quarter of the uridine and cytidine residues with 2-thio-U and 5-methyl-C, respectively, resulted in a significant reduction in toll-like receptor (TLR)-mediated recognition of the mRNA in mice. By reducing activation of the innate immune system, these modifications can therefore be used to effectively increase the stability and longevity of the mRNA in vivo; see, e.g., Konmann et al., supra.

It has also been shown that repeated administration of synthetic messaging RNAs incorporating modifications designed to bypass the innate antiviral response can reprogram differentiated human cells to pluripotency. See, e.g., Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Such modified mRNAs as primary reprogramming proteins can be an effective means of reprogramming a variety of human cell types. Such cells are called induced pluripotent stem cells (iPSCs) and it was found that enzymatically synthesized RNAs incorporating 5-methyl-CTP, pseudoUTP, and anti-reverse cap analog (ARCA) can be used to effectively evade the antiviral response of the cell; see, e.g., Warren et al., supra. Other modifications of nucleic acids described in this technology include, for example, the use of poly A tails, the addition of 5' cap analogs such as m7G(5')ppp(5')G (mCAP), modification of the 5' or 3' untranslated regions (UTRs), or treatment with phosphatases to remove 5' phosphates, and new methods are regularly developed.

Modifications that have been combined with RNA interference (RNAi), including small interfering RNA (siRNA), have developed a number of compositions and techniques suitable for generating modified RNA for use herein. siRNA presents particular challenges in vivo because its effects on gene silencing via mRNA interference are generally transient, which may require repeated administration. In addition, siRNA is double-stranded RNA (dsRNA) and mammalian cells have immune responses that have evolved to detect and neutralize dsRNA, which is often a byproduct of viral infection. Thus, there are mammalian enzymes such as PKR (dsRNA-responsive kinase) and potentially retinoic acid-inducible gene 1 (RIG-I) that mediate cellular responses to dsRNA, as well as toxin-like receptors such as TLR3, TLR7, and TLR8 that trigger induction of cytokines in response to such molecules; see, e.g., Angart et al., Pharmaceuticals (Basel) 6(4): 440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012); Burnett et al., Biotechnol J. 6(9): 1130-46 (2011); Judge and Maclachlan, Hum Gene Ther 19(2): 111-24. (2008); and a review of the references cited therein.

A variety of modifications have been developed and used to enhance RNA stability, reduce innate immune responses, and/or achieve other benefits that can be used in conjunction with introducing nucleic acids into human cells as described herein; see, e.g., Whitehead KA et al., Annual Review of Chemical and Biomolecular Engineering, 2:77-96 (2011); Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al., Curr Opin Mol Ther., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic Acid Chem, Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008): Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); review by Bremsen et al., Front Genet 3:154 (2012).

As described above, there are many commercial suppliers of modified RNA, many of which have specialized in modifications designed to improve the effectiveness of siRNA. Various methods are provided based on various findings reported in the literature. For example, Dharmacon indicates that replacement of non-bridging oxygens with sulfur (phosphothioate, PS) has been widely used to improve nuclease resistance of siRNA, as reported by Kale, Nature Reviews Drug Discovery 11: 125-140 (2012). Modification of the 2' position of the ribose has been reported to improve nuclease resistance of the internucleotide phosphate bond while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. Moderate PS backbone modifications in combination with small, well-tolerated 2'-substitutions (2'-O-, 2'-fluoro, 2'-hydrogen) have been associated with highly stable siRNAs for in vivo administration, as reported by Soutschek et al., Nature 432:173-178 (2004); and 2'-O-methyl modifications have been reported to be effective in improving stability, as reported by Volkov, Oligonucleotides 19:191-202 (2009). With respect to reducing the induction of innate immune responses, it has been reported that modification of specific sequences with 2'-O-methyl, 2'-fluoro, 2'-hydrogen can reduce TLR7/TLR8 interactions while generally retaining silencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505 (2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additional modifications (such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine) have also been shown to minimize immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko et al., Immunity 23:165-175 (2005).

As is also known in the art and commercially available, a number of conjugates can be applied to nucleic acids, such as RNA used herein, that can enhance their delivery and/or cellular uptake, including, for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers, and aptamers; see, for example, Winkler, Ther. Deliv. 4:791-809 (2013), and references cited therein for a review. 6.9. Carriers

The invention provides vectors comprising nucleic acids of the invention (e.g., as described in Section 6.8). In some embodiments, the nucleic acid comprises a nucleic acid encoding an engineered B-GEn polypeptide as described in Section 6.2. In some embodiments, the engineered B-GEn polypeptide coding sequence is codon-optimized, at least for the portion of the nuclease component encoding the engineered B-GEn polypeptide.

The vector (or nucleotide sequence) may further encode a gRNA.

In some embodiments, the vector comprising the nucleotide sequence may be an expression vector.

In some embodiments, the expression vector is a production vector of an engineered B-GEn polypeptide, for example, it can be used for expression/production of an engineered B-GEn polypeptide in a host cell. After expression/production of an engineered B-GEn polypeptide in a host cell, the engineered B-GEn polypeptide can be incorporated into RNP for nuclear transfection of a target cell.

Alternatively, the expression vector comprising the nucleotide sequence can be a delivery vector for an engineered B-GEn polypeptide, for example, it can be used to introduce the engineered B-GEn polypeptide coding sequence into a target cell intended for gene editing. After the expression/production of the engineered B-GEn polypeptide in the target cell, the engineered B-GEn polypeptide and the guide RNA molecule can edit the target cell. In some embodiments, the delivery vector further includes the coding sequence of the gRNA. In other embodiments, an independent nucleic acid encoding the gRNA is introduced into the target cell.

Contemplated expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses (e.g., Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for use in eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for use in eukaryotic cells include, but are not limited to, vectors pCTx-1, pCTx-2, and pCTx-3. Other vectors may be used as long as they are compatible with the desired host or target cell.

In some embodiments, the expression vector has one or more transcriptional and/or translational control elements. Depending on the expression cell/vector system used, any of a variety of suitable transcriptional and translational control elements (including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc.) can be used in the vector. The vector may also contain a ribosome binding site for initiation of translation and a transcriptional terminator.

Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that function in eukaryotic cells) include those from cytomegalovirus (CMV) (immediate early), herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, the human elongation factor-1 promoter (EF1), a hybrid construct with the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), the murine stem cell virus promoter (MSCV), the phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I.

In some embodiments, the promoter is an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). In some embodiments, the promoter is a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some embodiments, the promoter is a spatially restricted and/or temporally restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.). In some embodiments, the vector does not have a promoter for at least one gene intended to be expressed in the host cell if the gene would be expressed under an endogenous promoter present in the genome following its insertion into the genome.

For expression of small RNAs, including guide RNAs, various promoters, such as RNA polymerase III promoters, including, for example, U6 and H1, can be advantageous. Thus, such promoters can be advantageously incorporated into delivery vectors. Descriptions and parameters for enhancing the use of such promoters are known in the art, and additional information and methods are regularly described; see, for example, Ma, H. et al., Molecular Therapy - Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

In some embodiments, the vector is a self-deactivating vector that deactivates a viral sequence or component or other element of the CRISPR mechanism. Self-deactivating vectors are particularly useful for delivery vectors to select cells that retain the engineered B-GEn polypeptide coding sequence after gene editing is complete.

In some embodiments, the expression vectors are RNA vectors. In other embodiments, the expression vectors are DNA vectors. 6.9.1. RNA vectors

The expression vector of the present invention may be an RNA vector.

Particularly suitable vectors are viral replicons based on RNA viruses, such as alphaviruses and paramyxoviruses. Alphavirus and paramyxovirus replicons do not involve DNA intermediates for replication and therefore provide safer alternatives to several other commonly used viral vectors, including lentiviral and retroviral vectors (Yoshioka et al., 2013, Cell Stem Cell. 13(2):246-54; Yoshioka and Dowdy, 2017, PLOS ONE 12:e0182018). Alphaviruses are lipid-enveloped positive-sense RNA viruses that make up a genus of more than 30 viruses in the Togaviridae family, including Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), chikungunya (CHIK), Sindbis, Ross River, and O'nyong-nyong viruses, etc. Sendai viruses (SeV) are enveloped, single-stranded negative-sense paramyxoviruses that replicate episodically in the host cell cytoplasm.

Thus, in some embodiments, the RNA vector is derived from an RNA virus, such as an alphavirus, a paramyxovirus, a flavivirus, a rhabdovirus, a measles virus, or a picornavirus.

In some embodiments, the RNA vector is a single-stranded RNA replicon. In some embodiments, the single-stranded RNA replicon is a positive strand. In some other embodiments, the single-stranded RNA replicon is a negative strand. In some embodiments, the RNA vector comprises one or more coding sequences of one or more engineered B-GEn polypeptides and a self-replicating element.

The RNA replicon of the present invention generally includes a regulatory element, i.e., a subgenomic (SG) promoter, that can be operably linked to an engineered B-GEn polypeptide coding sequence. The sequence comprising the engineered B-GEn polypeptide coding sequence is generally flanked by 5' and 3' UTR sequences, and the 3' UTR sequence is generally followed by a polyadenylation signal.

The RNA vector construct can be generated from a DNA template (DNA plasmid construct). For example, the RNA construct can be transcribed from a DNA template using an SP6 or T7 in vitro transcription kit.

RNA vectors are particularly useful as delivery vectors. 6.9.2. DNA vectors

In some embodiments, the expression vector of the present invention is a DNA vector. The present invention provides two types of DNA vectors: (1) DNA vectors that are production vectors or delivery vectors and (2) DNA vectors that can be transcribed from RNA vectors of the present invention (as described in Section 6.9.1), as described in Section 6.9.2.2. DNA vectors of the present invention that can transcribe RNA replicas are sometimes referred to herein as "template vectors."

In some embodiments, the DNA vector of the present invention is a non-integrating DNA vector. For example, the vector can be an episomal vector. For example, many plasmids containing DNA viruses, such as adenovirus, simian vacuolating virus 40 (SV40), bovine papilloma virus (BPV) or budding yeast ARS (autonomous replication sequence) can be used without genomic integration.

In some embodiments, the DNA vector of the present invention includes a replication origin. Examples of replication origins that can be incorporated into the DNA vector of the present invention include the replication origin of lymphotropic herpes virus, gamma herpes virus, adenovirus, bovine papilloma virus, or yeast. In some embodiments, the replication origin is from a lymphotropic herpes virus or a gamma herpes virus corresponding to oriP of EBV, as a self-replicating element. In some embodiments, the lymphotropic herpes virus is Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS) or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gammaherpesviruses.

In certain embodiments, the vector of the present invention comprises the replication origin OriP of EBV. OriP is the site at or near the start of DNA replication and consists of two cis-acting sequences called repeat family (FR) and binary symmetry (DS) separated by about 1 kilobase pair. FR consists of 21 imperfect 30 bp repeat copies and contains 20 high-affinity EBNA-1 binding sites. When FR is bound by EBNA-1, it acts as a transcription enhancer for cis-acting promoters up to 10 kb in distance. DS is sufficient to initiate DNA synthesis in the presence of EBNA-1 and initiation occurs at or near the DS.

One or more of the expression cassettes in the replication DNA vector may further comprise a nucleotide sequence encoding a trans-acting factor that binds to the replication origin to replicate the extrachromosomal template. Alternatively or additionally, the somatic cell may express such a trans-acting factor.

In other embodiments, the DNA vectors of the present invention lack an origin of replication.

The DNA vectors of the present invention typically comprise one or more promoters SP6 or T7 to drive expression of the engineered B-GEn polypeptide in the case of a DNA vector intended to be a production vector or to drive expression of an RNA replicon in the case of a DNA vector intended to be a template vector. 6.9.2.1. Expression vectors

In some embodiments, the expression vector is a DNA vector comprising an expression cassette for expressing one or more proteins of interest, which can be operably linked to a regulatory element comprising a promoter suitable for driving expression of the engineered B-GEn polypeptide in a cell type of interest. Examples of promoters suitable for driving expression of proteins in mammalian cells include the cytomegalovirus (CMV) promoter, the EF1a promoter, the SV40 promoter, the Ubc promoter, the human beta actin promoter, the PGK1 promoter, and the CAG promoter.

DNA vectors used to directly express engineered B-GEn polypeptides (rather than serving as templates for expression of RNA vectors as described in Section 6.9.2.2) need not include RNA replicon self-replicating sequences, such as the nsP1-nsP4 proteins of VEEV or the NP, P, and L proteins of Sendai virus.

In some embodiments, the DNA expression vector is a non-replicating DNA vector. In some embodiments, the DNA expression vector is a replicating DNA vector. 6.9.2.2. Template vector

The DNA vectors of the present invention can also be used as templates for transcription of RNA replicons as described herein. Thus, the "expression cassette" included in the template vector is intended to be transcribed from the RNA replicons produced by transcription of the RNA replicons.

Thus, the template vector of the present invention comprises a nucleotide sequence encoding an RNA replicon as described herein under the control of a regulatory element (eg, an SP6 or T7 promoter).

In some embodiments, the template DNA vector is a non-replicating DNA vector. In some embodiments, the template DNA vector is a replicating DNA vector.

In some embodiments, the template vectors are used for in vitro transcription of RNA replicons, which are then introduced into cells to drive expression of engineered B-GEn polypeptides. 6.9.3. Viral vectors

Recombinant adeno-associated virus (AAV) vectors may be used for delivery. Techniques known in the art for producing rAAV particles are to provide cells with the polynucleotide to be delivered between two AAV inverted terminal repeats (ITRs), the AAV rep and cap genes, and helper viral functions. The production of rAAV requires the presence of the following components within a single cell (represented herein as a packaging cell): the polynucleotide of interest between the two ITRs, the AAV rep and cap genes separated from (i.e., not present in) the AAV genomic components, and helper viral functions. The AAV rep and cap genes can be from any AAV serotype from which recombinant virus can be derived and can be from an AAV serotype different from the ITRs on the packaging polynucleotide, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh. 74. The generation of pseudotyped rAAVs is disclosed, for example, in WO 01/83692. AAV serotypes Genbank registration number AAV-1 NC_002077.1 AAV-2 NC_001401.2 AAV-3 NC_001729.1 AAV-38 AF028705.1 AAV-4 NC_001829.1 AAV-5 NC_006152.1 AAV-6 AF028704.1 AAV-7 NC_006260.1 AAV-8 NC_006261.1 AAV-9 AX753250.1 AAV-10 AY631965.1 AAV-11 AY631966.1 AAV-12 00813647.1 AAV-13 EU285562.1

One method of generating packaging cells is to establish a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or plasmids) comprising the polynucleotide of interest between the AAV ITRs, the AAV rep and cap genes isolated from the AAV genome, and a selectable marker (such as a neomycin resistance gene) is integrated into the genome of the cell. The AAV genome has been introduced into bacterioplasts by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or bacilli rather than plasmids to introduce the rAAV genome and/or the rep and cap genes into packaging cells.

The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and lmmunol., 158:97-129). Various methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); Mclaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173,414; WO 95/13365 and corresponding U.S. Patent No. 5,658.776; WO 95/13392; WO WO97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595.

The AAV vector serotype used for transduction depends on the target cell type. For example, the following exemplary cell types are known to be transduced by the indicated AAV serotypes, etc. Tissue / cell type Serotype Liver AAV8, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, AAV9 Central nervous system AAV5, AAV1, AAV4 RPE AAV5, AAV4 Photoreceptor cells AAV5 lung AAV9 Heart AAV8 Pancreas AAV8 Kidney AAV2

Many suitable expression vectors are known in the art and many are commercially available. The following vectors are provided by way of example; for use with eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used so long as it is compatible with the host cell. 6.10. Host Cells and Recombinant Expression

In some embodiments, host cells can be used to express gRNA, sgRNA or the engineered B-GEn polypeptide of the present invention. Suitable host cells include naturally occurring cells; genetically modified cells (e.g., cells genetically modified in the laboratory), and cells manipulated in vitro in any manner. In some embodiments, the host cell is isolated.

The host cell can be eukaryotic or prokaryotic and includes, for example, yeast (such as Pichia pastoris or Saccharomyces cerevisiae), bacteria (such as Escherichia coli or Bacillus subtilis), insect Sf9 cells (such as SF9 cells infected with bacillus virus), or mammalian cells (such as human embryonic kidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293 cells, and monkey COS-7 cells).

The host cell may be from an established cell line, or it may be a primary cell, wherein "primary cell," "primary cell line," and "primary culture" are used interchangeably herein to refer to cells and cell cultures that have been derived from an individual and allowed to grow in vitro to achieve a limited number of passages (e.g., divisions) of the culture. For example, a primary culture includes a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough times to pass the crisis phase. A primary cell line may be maintained in vitro for less than 10 passages. In some embodiments, the host cell is a PSC (e.g., an iPSC or an ESC), or a PSC-derived cell (e.g., a PSC-derived neuron, a PSC-derived microglia, a PSC-derived cardiomyocyte, a PSC-derived cell of the eye).

If the cells are primary cells, such cells may be harvested from an individual by any suitable method. Suitable solutions may be used to disperse or suspend the harvested cells. The harvested cells may be used immediately, or they may be stored, frozen, for a long period of time, thawed and capable of being reused. In such cases, the cells are generally frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner commonly known in the art for thawing frozen cultured cells. 6.11. Target cells

In some embodiments, the engineered V-type endonuclease or B-GEn CRISPR-Cas system is introduced into a target cell or a population of target cells. Methods for introducing proteins and nucleic acids into target cells are further described in Section 6.12.

The target cells and target cell populations of the present invention may be cells in which gene editing by the system of the present invention has occurred, or cells in which components of the system of the present invention have been introduced or expressed but gene editing has not yet occurred, or a combination thereof. In various embodiments, the cell population may include, for example, a population in which at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells have been gene edited by the system of the present invention.

In some embodiments, the methods of the present invention can be used for transcriptional regulation in cells that induce mitosis or post-mitosis in vivo and/or in vitro and/or in vitro. In some embodiments, the methods of the present invention can be used for DNA cleavage, DNA modification, and/or transcriptional regulation in cells that induce mitosis or post-mitosis in vivo and/or in vitro and/or in vitro (e.g., to produce genetically modified cells that can be reintroduced into an individual).

Since the guide RNA provides specificity by hybridizing to the target DNA, the mitotic and/or post-mitotic cells can be any of a variety of target cells and modified in vivo or in vitro. Suitable target cells include, but are not limited to, bacterial cells; archaeal cells; unicellular eukaryotic organisms; plant cells; algal cells, such as Botryococcus braunii , Chlamydomonas reinhardtii , Nannochloropsis gaditana , Chlorella pyrenoidosa , Sargassum patens , C. agardh , and the like; fungal cells; animal cells; cells from invertebrates (e.g., insects, cnidarians, echinops, nematodes, etc.); eukaryotic parasites (e.g., malarial protozoa); parasite, such as Plasmodium falciparum ; worms, etc.); cells from vertebrates (e.g., fish , amphibians, reptiles, birds, mammals); mammalian cells, such as rodent cells, human cells, non-human primates, etc. In some embodiments, the target cell can be any human cell. Suitable target cells include naturally occurring cells; genetically modified cells (e.g., cells genetically modified in the laboratory, such as by "human hands"), and cells manipulated in vitro in any manner. In some embodiments, the target cell is isolated.

Any type of cell can be of interest as a host cell or target cell for modification in vivo or in vitro. In various embodiments, the host cell or target cell is a stem cell (e.g., PSC, such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSC)), a germ cell; a somatic cell, such as a fibroblast, a hematopoietic cell, an immune cell (e.g., a T-lymphocyte, a B-lymphocyte, a dendritic cell, or a macrophage), a neuron, a muscle cell, a bone cell, a liver cell, a pancreatic cell; an embryonic cell at any stage (e.g., a zebrafish embryo at the 1-cell, 2-cell, 4-cell, 8-cell, etc. stage; etc.). The cell may be from an established cell line, or it may be a primary cell, wherein "primary cell," "primary cell line," and "primary culture" are used interchangeably herein to refer to cells and cell cultures that have been derived from an individual and allowed to grow in vitro to achieve a limited number of passages (e.g., divisions) of the culture. For example, a primary culture includes a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough times to pass a crisis phase. A primary cell line may be maintained in vitro for less than 10 passages. In some embodiments, the target cell is a single cell organism, or is grown in culture. In some embodiments, the host cell is the same as the target cell. In some embodiments, the target cell is modified to become another cell type so that the resulting host cell is different from the target cell. As an example, the target cell can be a PSC (e.g., an iPSC), which is then differentiated into a PSC-derived cell (e.g., a PSC-derived neuron) so that the host cell is a neuron. Alternatively, the target cell is not a PSC, but is subsequently dedifferentiated or reprogrammed to a PSC.

If the cells are primary cells, they can be harvested from an individual by any suitable method. For example, leukocytes can be suitably harvested by hematopheresis, leukocyte separation, density gradient separation, etc., and cells from tissues (such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc.) are most suitably harvested by biopsy. The harvested cells can be dispersed or suspended using a suitable solution. Such a solution is generally a balanced salt solution, such as physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc., which is appropriately supplemented with fetal bovine serum or other naturally occurring factors, combined with a low concentration (e.g., 5 to 25 mM) of an acceptable buffer. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately, or they can be stored, frozen, for a long period of time, thawed and can be reused. In such cases, the cells are generally frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner commonly known in the art for thawing frozen cultured cells.

In some embodiments, the target cell is located in an individual and the methods of the invention include administering a B-GEn CRISPR-Cas system (e.g., a ribonucleoprotein comprising an engineered B-GEn polypeptide of the invention) to an individual (e.g., a mammalian individual, such as a human or livestock individual). Exemplary in vivo cells to which the B-GEn CRISPR-Cas system can be delivered include, but are not limited to, fibroblasts, hematopoietic cells, immune cells (e.g., T-lymphocytes, B-lymphocytes, dendritic cells, or macrophages), neurons, neuroglia, muscle cells (e.g., cardiomyocytes), bone cells, hepatocytes, and pancreatic cells. 6.11.1. Pluripotent stem cells (PSC)

In some embodiments, the target cells are stem cells, for example, particularly pluripotent stem cells (PSCs), such as induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs), which can be differentiated and used to generate large numbers of specific cell types that can be delivered for regenerative medicine in patients with many different diseases. In the case of PSCs, differentiation is a process of lineage specification and can be achieved using cell-specific protocols.

After modifying PSC (e.g., hESC or iPSC) by introducing an engineered V-type endonuclease or B-GEn polypeptide of the present invention, the PSC can be differentiated into a cell type of interest for cell therapy. In some embodiments, the genome of the PSC is edited by an engineered B-GEn polypeptide of the present invention prior to differentiation.

PSCs (e.g., PSCs comprising an engineered V-type endonuclease or B-GEn polypeptide of the invention or whose genomes have been edited by an engineered V-type endonuclease or B-GEn polypeptide of the invention) can be differentiated into cells suitable for therapy, including cells in the endoderm (e.g., lung, thyroid or pancreatic cells or their precursors), ectoderm (e.g., skin, neuron or pigment cells or their precursors), and mesoderm (e.g., heart cells, skeletal muscle cells, red blood cells, smooth muscle cells or their precursors or progenitors) lineages.

In some embodiments, the PSCs of the present invention are differentiated into cardiac cells. In various embodiments, the cardiac cells are cardiac progenitor cells or mature or immature (atrial or ventricular) cardiomyocytes.

In other embodiments, the PSCs of the present invention are differentiated into oligodendrocyte precursor cells or oligodendrocytes.

In other embodiments, the PSCs of the present invention are differentiated into neural lineage cells, such as neural crest cells, astrocytes, dopamine neuron precursor cells, dopamine neuron cells, midbrain dopamine neuron precursor cells, midbrain dopamine neurons, true midbrain dopamine (DA) neurons, dopamine neuron precursor cells, floor plate midbrain precursor cells, floor plate midbrain DA neurons.

In other embodiments, the PSCs of the present invention are differentiated into photoreceptor cells, photoreceptor progenitor cells, retinal pigment epithelial cells, neural retinal cells, or neural retinal progenitor cells.

In some embodiments, the PSCs of the present invention are differentiated into microglia or microglia precursor cells.

In some embodiments, the PSCs of the present invention are differentiated into macrophages.

In some embodiments, the PSCs of the present invention are differentiated into intestinal progenitor cells or intestinal cells.

In some embodiments, the PSCs of the present invention are differentiated into immune cells, such as T lymphocytes, B lymphocytes, dendritic cells or macrophages.

In some embodiments, the PSCs can be genetically engineered prior to differentiation into the cell type of interest (e.g., to produce a functional protein that is deficient in the patient, to produce a therapeutic protein, including an off switch, or to evade immune detection to support allogeneic applications). 6.12. Methods of introducing nucleic acids into host and target cells

In some embodiments, the methods of the present invention include introducing one or more nucleic acids comprising a nucleotide sequence encoding a guide RNA and/or a nucleotide sequence encoding an engineered B-GEn polypeptide (e.g., a codon-optimized nucleotide sequence) into a host or target cell (or a host or target cell population). In some embodiments, the methods of the present invention include introducing a guide RNA and/or a nucleotide sequence encoding an engineered B-GEn polypeptide (e.g., a codon-optimized nucleotide sequence) into a host or target cell (or a host or target cell population).

In some embodiments, the target cell (e.g., a cell comprising DNA targeted by a guide RNA to be edited by an engineered B-GEn polypeptide) is in vitro, such as in cell culture. In some embodiments, the target cell is an in vivo cell in an individual (e.g., a mammal, such as a human) for whom gene therapy is intended.

In some embodiments, the nucleotide sequence encoding the guide RNA and/or the engineered B-GEn polypeptide can be operably linked to an inducible promoter. In some embodiments, the nucleotide sequence encoding the guide RNA and/or the engineered B-GEn polypeptide can be operably linked to a constitutive promoter.

A guide RNA, or a nucleic acid comprising a nucleotide sequence encoding the guide RNA, can be introduced into a host or target cell by any of a variety of well-known methods. Similarly, in cases where the method involves introducing a nucleic acid comprising a nucleotide sequence encoding an engineered B-GEn polypeptide (e.g., a codon-optimized nucleotide sequence) into a host or target cell, such a nucleic acid can be introduced into a host or target cell by any of a variety of well-known methods. A guide nucleic acid (RNA or DNA; e.g., a guide RNA or one or more DNA molecules encoding the guide RNA) and/or an engineered B-GEn polypeptide encoding nucleic acid (RNA or DNA) can be delivered by viral or non-viral delivery vectors known in the art.

Methods for introducing nucleic acids into host or target cells are known in the art, and any known method can be used to introduce nucleic acids (e.g., expression constructs) into stem cells or pioneer cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-polydextrose-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: 50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like, including but not limited to exosome delivery.

The polynucleotide sequence (e.g., a) can be delivered by non-viral delivery vehicles (including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes). Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses on non-viral delivery vehicles for siRNA, which can also be used to deliver other nucleic acids).

Suitable systems and techniques for delivering nucleic acids (e.g., mRNA and sgRNA) of the present invention for gene editing include lipid nanoparticles (LNPs). As used herein, the term "plastid nanoparticles" includes liposomes, regardless of their lamellarity, shape or structure, and lipoplexes used to introduce nucleic acids and/or polypeptides into cells as described. Such lipid nanoparticles can be complexed with biologically active compounds (e.g., nucleic acids and/or polypeptides) and can be used as in vivo delivery vehicles. In general, any method known in the art can be applied to prepare lipid nanoparticles containing one or more nucleic acids of the present invention and to prepare complexes of biologically active compounds and such lipid nanoparticles. Examples of such methods are widely disclosed in, for example, Biochim Biophys Acta 1979, 557:9; Biochim et Biophys Acta 1980, 601:559; Liposomes: A practical approach (Oxford University Press, 1990); Pharmaceutica Acta Helvetiae 1995, 70:95; Current Science 1995, 68:715; Pakistan Journal of Pharmaceutical Sciences 1996, 19:65; Methods in Enzymology 2009, 464:343). Particularly suitable systems and technologies for preparing LNP formulations comprising one or more nucleic acids and/or polypeptides of the invention include, but are not limited to, those developed by Intellia (see, e.g., WO2017173054A1), Alnylam (see, e.g., WO2014008334A1), Modernatx (see, e.g., WO2017070622A1 and WO2017099823A1), TranslateBio, Acuitas (see, e.g., WO2018081480A1), Genevan Sciences, Arbutus Biopharma, Tekmira, Arcturus, Merck (see, e.g., WO2015130584A2), Novartis (see, e.g., WO2015095340A1), and Dicerna; each of which is incorporated herein by reference in its entirety.

Suitable nucleic acids comprising nucleotide sequences encoding engineered B-GEn polypeptides and/or guide RNAs include expression vectors. In some embodiments, the expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, e.g., U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, etc. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; polio virus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543-2549, 1994; Borras et al., Gene Ther 6:515-524, 1999; Li and Davidson, PNAS 92:7700-7704, 1995; Sakamoto et al., H Gene Ther 5:1088-1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683-690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Viral. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319-23, 1997; Takahashi et al., J Virol 73:7812-7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses (e.g., Rous sarcoma virus, Harvey Sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus)); and the like.

In some embodiments, B-GEn ribonucleoprotein comprising B-GEn endonuclease and sgRNA is delivered to target cells by nucleofection, a method of creating transient pores in the cell membrane by delivering nucleic acids to cells using cell-specific reagents and electrical parameters.

The engineered B-GEn V-type CRISPR-Cas system can be delivered to target cells by delivery vectors such as viral vectors. The engineered B-GEn V-type CRISPR-Cas system can also be delivered to target cells by non-viral delivery vectors, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Some exemplary non-viral delivery vectors are described in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011).

In some embodiments, the engineered B-GEn V-type CRISPR-Cas system is delivered to a target cell using a delivery vector as described in Section 6.9. 6.13. Gene Editing Methods

The present invention also provides methods for gene editing using engineered V-type endonucleases or B-GEn polypeptide systems. Such gene editing methods may include methods for targeting, editing, modifying or manipulating target DNA at one or more locations in the genome of a host or target cell (or multiple hosts or target cells) (whether in vitro, ex vivo or in vivo) or in a cell-free environment. Generally, the gene editing methods include systematically introducing an engineered V-type endonuclease or B-GEn polypeptide into a host or target cell (or a host or target cell population) or a target DNA sequence comprising a cell-free environment under conditions suitable for the engineered V-type endonuclease or B-GEn polypeptide to perform one or more modifications (e.g., nicking or cleavage or base editing) in the target DNA, wherein the engineered V-type endonuclease or B-GEn polypeptide is guided to the target DNA by a guide RNA in a treated or untreated form.

The gene editing method may include introducing the engineered V-type nuclease or B-GEn polypeptide system of the present invention into a host or target cell (or a host or target cell population) as an RNP complex and/or introducing one or more nucleic acids comprising a guide RNA or a nucleotide sequence encoding a guide RNA and a nucleotide sequence encoding an engineered V-type nuclease or B-GEn polypeptide (e.g., a codon-optimized nucleotide sequence) into a host or target cell (or a host or target cell population) (e.g., via a virus (such as AAV) or via LNP). The engineered V-type endonuclease or B-GEn polypeptide system of the present invention (e.g., in the form of an RNP complex; one or more nucleic acid molecules encoding a guide RNA and one or more nucleotide sequences encoding an engineered V-type endonuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences); or a guide RNA and one or more nucleotide sequences encoding an engineered V-type endonuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences)) can be introduced into a host or target cell (or a host or target cell population) by any of a variety of well-known viral or non-viral delivery methods. Such gene editing methods can be used in vivo, in vitro, or in vitro on a host or target cell (or a host or target cell population).

In some embodiments, the gene editing method includes introducing an engineered V-type nuclease or B-GEn polypeptide system of the present invention (e.g., in the form of an RNP complex; one or more nucleic acid molecules encoding a guide RNA and one or more nucleotide sequences encoding an engineered V-type nuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences); or a guide RNA and one or more nucleotide sequences encoding an engineered V-type nuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences)) into an individual (e.g., a mammalian individual, such as a human individual) in which gene editing is desired (e.g., for gene therapy purposes) to perform one or more modifications (e.g., nicking or cutting or base editing) in the target DNA in vivo.

In some embodiments, the gene editing method includes introducing an engineered V-type nuclease or B-GEn polypeptide system of the present invention (e.g., in the form of an RNP complex; one or more nucleic acid molecules encoding a guide RNA and one or more nucleotide sequences encoding an engineered V-type nuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences); or a guide RNA and one or more nucleotide sequences encoding an engineered V-type nuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences)) to perform one or more modifications (e.g., nicking or cutting or base editing) in an isolated target DNA.

In some embodiments, the gene editing method includes introducing an engineered V-type nuclease or B-GEn polypeptide system of the present invention (e.g., in the form of an RNP complex; one or more nucleic acid molecules encoding a guide RNA and one or more nucleotide sequences encoding an engineered V-type nuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences); or a guide RNA and one or more nucleotide sequences encoding an engineered V-type nuclease or B-GEn polypeptide (e.g., one or more codon-optimized nucleotide sequences)) to perform one or more modifications (e.g., nicking or cutting or base editing) in the target DNA in vitro.

In some embodiments, the gene editing method includes contacting a cell with an engineered V-type endonuclease or B-GEn polypeptide system in the form of an RNP complex comprising an engineered V-type or B-GEn polypeptide and a guide RNA. Exemplary details of the preparation, composition, and delivery of RNP complexes are described in Section 6.7. In some embodiments, RNP complexes can also be delivered to the cell using a delivery method that increases the porosity of the plasma membrane of the cell, such as by electroporation or nuclear transfection.

In some embodiments, the gene editing method comprises contacting a cell with an engineered V-type endonuclease or B-GEn polypeptide system in the form of an LNP (e.g., an LNP comprising an RNP (e.g., an LNP as described in the previous paragraph) or an LNP comprising a nucleic acid encoding an engineered V-type endonuclease or B-GEn polypeptide and a guide RNA or a nucleic acid encoding the guide RNA). Any method known in the art can be applied to prepare the LNP, for example, as described in Section 6.12.

In some embodiments, the gene editing method comprises contacting a cell with an engineered V-type endonuclease or B-GEn polypeptide system in the form of one or more viruses (e.g., one or more AAVs, whose genomes comprise one or more nucleotide sequences encoding an engineered V-type endonuclease or B-GEn polypeptide and a nucleic acid encoding a guide RNA). The use of viruses to introduce transgenes (e.g., nucleic acids encoding an engineered V-type endonuclease or B-GEn polypeptide and guide RNA encoding sequences) is known in the art and is described in Section 6.9.3. Other methods and delivery vectors for introducing nucleic acids encoding engineered V-type endonucleases or B-GEn polypeptides or guide RNAs are described in Sections 6.9 and 6.12. 6.14. Pharmaceutical Compositions

Also disclosed herein are pharmaceutical formulations and agents comprising the B-GEn protein, gRNA, nucleic acid or multiple nucleic acids, system, particle or multiple particles of the present invention and a pharmaceutically acceptable formulation.

Suitable formulations include, but are not limited to, salts, diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, adsorbents, solvents, pH regulators, antioxidants, anti-infective agents, suspending agents, wetting agents, viscosity regulators, tonic agents, stabilizers, and other components and combinations thereof. Suitable pharmaceutically acceptable formulations can be selected from materials that are generally recognized as safe (GRAS) and can be administered to an individual without causing undesirable biological side effects or unwanted interactions. Suitable excipients and their formulations are described in Remington's Pharmaceutical Sciences, 16th edition, 1980, Mack Publishing Co. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymer compounds (such as polyacetic acid, polyglycolic acid, hydrogels, etc.), or incorporated into liposomes, microemulsions, micelles, monolayer or multilayer vesicles, erythrocyte ghosts or spheroidal blasts. Suitable dosage forms for administration (e.g., parenteral administration) include solutions, suspensions, and emulsions.

The components of the pharmaceutical formulation can be dissolved or suspended in a suitable solvent such as water, Ringer's solution, phosphate buffered saline (PBS) or isotonic sodium chloride. The formulation can also be a sterile solution, suspension or emulsion in a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.

In some cases, the formulation may include one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and include glycerin, mannitol, sorbitol, sodium chlorate and other electrolytes. In some cases, the formulations may be buffered with an effective amount of a buffer required to maintain a pH suitable for parenteral administration. Some examples of suitable buffers that are well known to those skilled in the art and that are useful are acetate, borate, carbonate, citrate and phosphate buffers.

In some embodiments, the formulation may be distributed or packaged in liquid form, or alternatively as a solid obtained, for example, by lyophilizing a suitable liquid formulation, which may be reconstituted with a suitable carrier or diluent prior to administration. In some embodiments, the formulations may comprise a guide RNA and a type II Cas protein in a pharmaceutically effective amount sufficient to edit a gene in a cell. The pharmaceutical compositions may be formulated for medical and/or veterinary use.

In some embodiments, the B-GEn endonuclease complexes can be introduced into host cells (e.g., iPSCs) to produce genetically modified cells that can be reintroduced into an individual. The iPSC-derived cells described herein can be provided in a pharmaceutical composition containing the cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a cell culture medium that does not contain any animal-derived components as needed. For storage and transportation, the cells can be frozen at < -70°C (e.g., on dry ice or in liquid nitrogen). Prior to use, the cells can be thawed and diluted in a sterile cell culture medium that supports the cell type of interest.

The cells can be administered to the patient systemically (e.g., by intravenous injection or infusion) or locally (e.g., by direct injection into local tissues, such as the heart, brain, and sites of damaged tissue). Various methods are known in the art for administering cells to tissues or organs of a patient, including, but not limited to, intracoronary administration, intramyocardial administration, endocardial administration, or intracranial administration.

A therapeutically effective number of iPSC-derived cells is administered to a patient. As used herein, the term "therapeutically effective" refers to a number of cells or an amount of a pharmaceutical composition that, when administered to a human subject suffering from or susceptible to a disease, disorder, and/or condition, is sufficient to treat, prevent, and/or delay the onset or progression of the disease, disorder, and/or condition. A person of ordinary skill will appreciate that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose. In some embodiments, at least 10 ³ (e.g., at least 10 ⁴ , at least 10 ⁵ , at least 10 ⁶ , at least 10 7, at least 10 ⁸ , at least 10 ⁹ , at least ^{10 10} , at least 10 ¹¹ , or at least 10 ¹² ) cells are administered to a subject at one time in one or ^{more sites} . In some embodiments, 10 ³ to 10 ¹⁸ (e.g., 10 ³ to 10 ⁴ , 10 3 to 10 5, 10 3 to 10 ⁶ , 10 ³ to 10 7, 10 ³ to 10 ⁸ , 10 ³ to 10 ⁹ , 10 ³ to 10 ¹⁰ , 10 ³ to 10 ¹¹ , 10 ³ to 10 ¹² , ^{10 6} to 10 ⁷ , 10 ⁶ to 10 ⁸ , 10 ⁶ to 10 ⁹ , 10 ⁶ to 10 ¹⁰ , 10 ⁶ to 10 ¹¹ , 10 ⁶ to 10 ¹² , 10 ⁹ to 10 ¹⁰ , ^{10 9 to} 10 ¹¹ , ^{10 9} to 10 ¹² ) cells are administered to an individual at one time in ^{one or} more sites. In some embodiments, more than 10 ¹² (e.g., more than 10 ¹² , more than 10 ¹³ , more than 10 ¹⁴ , more than 10 15, more than 10 ¹⁶ , more than 10 ¹⁷ , more than 10 ¹⁸ , or more) cells are administered to an individual at ^one time at one or more sites. 7. Numbered Embodiments

Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of the invention. The present invention is illustrated by the numbered embodiments described below. Unless otherwise specified, the features of any concept, aspect and/or embodiment described in the above detailed description can be applied to any of the following numbered embodiments. 1. A polypeptide comprising an amino acid other than aspartic acid at a position corresponding to D504 of SEQ ID NO: 1 (B-GEn.1) or D501 of SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2). 2. A polypeptide as in Example 1, which is an engineered V-type nuclease polypeptide. 3. The polypeptide of embodiment 2, which is an engineered Bacillusales type V endonuclease polypeptide. 4. The polypeptide of any one of embodiments 1 to 3, which is a B-GEn polypeptide. 5. The polypeptide of any one of embodiments 1 to 4, which has an arginine at a position corresponding to D504 of SEQ ID NO: 1 (B-GEn.1) or D501 of SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2). 6. The polypeptide of any one of embodiments 1 to 5, which comprises a target interaction sequence motif of any one of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203; and SEQ ID NO: 204. 7. The polypeptide of any one of embodiments 1 to 6, comprising a RuvC I domain comprising an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11. 8. The polypeptide of embodiment 7, wherein the RuvC I domain comprises an amino acid sequence having at least 40% sequence identity to the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11. 9. The polypeptide of embodiment 7, wherein the RuvC I domain comprises an amino acid sequence having at least 70% sequence identity to the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11. 10. The polypeptide of embodiment 7, wherein the RuvC I domain comprises an amino acid sequence having at least 80% sequence identity to the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11. 11. The polypeptide of embodiment 7, wherein the RuvC I domain comprises an amino acid sequence having at least 90% sequence identity with the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11. 12. The polypeptide of any one of embodiments 1 to 11, comprising a RuvC II domain comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12. 13. The polypeptide of embodiment 12, wherein the RuvC II domain comprises an amino acid sequence having at least 60% sequence identity with the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12. 14. The polypeptide of embodiment 12, wherein the RuvC II domain comprises an amino acid sequence having at least 70% sequence identity with the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12. 15. The polypeptide of embodiment 12, wherein the RuvC II domain comprises an amino acid sequence having at least 80% sequence identity with the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12. 16. The polypeptide of embodiment 12, wherein the RuvC II domain comprises an amino acid sequence having at least 90% sequence identity with the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12. 17. The polypeptide of any one of embodiments 1 to 16, comprising a RuvC III domain comprising an amino acid sequence having at least 80%, at least 85% or at least 90% sequence identity with the RuvC III domain of SEQ ID NO: 10 or SEQ ID NO: 13. 18. The polypeptide of embodiment 17, wherein the RuvC III domain comprises an amino acid sequence having at least 80% sequence identity with the RuvC III domain of SEQ ID NO: 10 or SEQ ID NO: 13. 19. The polypeptide of embodiment 17, wherein the RuvC III domain comprises an amino acid sequence having at least 85% sequence identity with the RuvC III domain of SEQ ID NO: 10 or SEQ ID NO: 13. 20. The polypeptide of embodiment 17, wherein the RuvC III domain comprises an amino acid sequence having at least 90% sequence identity with the RuvC III domain of SEQ ID NO: 10 or SEQ ID NO: 13. 21. The polypeptide of any one of embodiments 1 to 20, comprising the amino acid sequence of SEQ ID NO: 8. 22. The polypeptide of any one of embodiments 1 to 21, comprising the amino acid sequence of SEQ ID NO: 9. 23. The polypeptide of any one of embodiments 1 to 22, comprising the amino acid sequence of SEQ ID NO: 10. 24. The polypeptide of any one of embodiments 1 to 20, comprising the amino acid sequence of SEQ ID NO: 11. 25. The polypeptide of any one of embodiments 1 to 20 and 24, comprising the amino acid sequence of SEQ ID NO: 12. 26. The polypeptide of any one of embodiments 1 to 20 and 24 to 25, comprising the amino acid sequence of SEQ ID NO: 13. 27. A polypeptide, which is optionally a polypeptide as in any one of embodiments 1 to 26, comprising the following amino acid sequence: (a) having a substitution at a position corresponding to D501 of SEQ ID NO: 3, wherein the substitution: (i) is a substitution D501R compared to the amino acid sequence of SEQ ID NO: 3; or (ii) increases gene editing efficiency activity compared to the corresponding amino acid sequence without the substitution at position D501; and (b) having: (i) at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 1 (B-GEn.1), SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2); (ii) (A) SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203; and SEQ ID NO: 3 (B) a RuvC I domain comprising an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to the RuvC I domain of SEQ ID NO: 8 or SEQ ID NO: 11; (C) a RuvC II domain comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to the RuvC II domain of SEQ ID NO: 9 or SEQ ID NO: 12; (D) a RuvC III domain comprising an amino acid sequence having at least 80%, at least 85% or at least 90% sequence identity to the RuvC III domain of SEQ ID NO: 10 or SEQ ID NO: 13; or (E) any combination of two, three or all four of (A), (B), (C) and (D); (iii) Up to 25 amino acid insertions, substitutions and/or deletions compared to the amino acid sequence of SEQ ID NO: 1 (B-GEn.1), SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2); (iv) (b)(i) and (b)(ii); (v) (b)(i) and (b)(iii); (vi) (b)(ii) and (b)(iii); or (vii) (b)(i), (b)(ii) and (b)(iii). 28. The polypeptide of any one of embodiments 1 to 27, which has a gene editing efficiency that is at least 50% higher than the gene editing efficiency of a corresponding polypeptide lacking an amino acid substitution at a position corresponding to D504 of SEQ ID NO: 1 (B-GEn. 1) or SEQ ID NO: 2 (B-GEn. 1.2) or SEQ ID NO: 3 (B-GEn. 2). 29. The polypeptide of any one of embodiments 1 to 27, which has a gene editing efficiency that is at least 70% higher than the gene editing efficiency of a corresponding polypeptide lacking an amino acid substitution at a position corresponding to D504 of SEQ ID NO: 1 (B-GEn. 1) or SEQ ID NO: 2 (B-GEn. 1.2) or SEQ ID NO: 3 (B-GEn. 2). 30. The polypeptide of any one of embodiments 1 to 27, which has a gene editing efficiency that is at least 90% higher than the gene editing efficiency of the corresponding polypeptide lacking the amino acid substitution at the position corresponding to D504 of SEQ ID NO: 1 (B-GEn.1) or SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2). 31. The polypeptide of any one of embodiments 28 to 30, wherein the gene editing efficiency is assessed by an in-cell gene editing assay, optionally, wherein the gene editing assay is as described in Example 1. 32. The polypeptide of any one of embodiments 27 to 31, wherein the sequence identity is relative to SEQ ID NO: 1 and the amino acid insertions, substitutions and/or deletions are relative to SEQ ID NO: 1. 33. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. 34. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. 35. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 1. 36. The polypeptide of any one of embodiments 27 to 31, wherein the sequence identity is relative to SEQ ID NO: 2 and the amino acid insertions, substitutions and/or deletions are relative to SEQ ID NO: 2. 37. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2. 38. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. 39. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 2. 40. The polypeptide of any one of embodiments 27 to 31, wherein the sequence identity is relative to SEQ ID NO: 3 and the amino acid insertions, substitutions and/or deletions are relative to SEQ ID NO: 3. 41. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 3. 42. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. 43. The polypeptide of embodiment 32, wherein the amino acid sequence has at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 3. 44. A polypeptide comprising the amino acid sequence of SEQ ID NO: 4. 45. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5. 46. A polypeptide comprising the amino acid sequence of SEQ ID NO: 6. 47. The polypeptide of any one of embodiments 1 to 46, further comprising at least one nuclear localization signal ("NLS"). 48. The polypeptide of embodiment 47, comprising at least one NLS located at the C-terminus of the amino acid sequence, optionally wherein: (a) the polypeptide lacks any NLS located at the N-terminus of the amino acid sequence; or (b) the polypeptide comprises at least one NLS located at the N-terminus of the amino acid sequence. 49. The polypeptide of embodiment 47 or 48, lacks any NLS located at the N-terminus of the amino acid sequence. 50. The polypeptide of any one of embodiments 47 to 49, comprising a linker sequence between the amino acid sequence and each NLS. 51. The polypeptide of any one of embodiments 47 to 50, wherein each NLS comprises an amino acid sequence independently selected from the NLS sequences described in Table 2. 52. The polypeptide of any one of Examples 47 to 51, wherein each linker sequence is independently selected from the linker sequences described in Table 3. 53. A nucleic acid comprising a nucleotide sequence encoding a polypeptide of any one of Examples 1 to 52. 54. The nucleic acid of Example 53, wherein the nucleotide sequence encoding a polypeptide of any one of Examples 1 to 52 can be operably linked to a promoter. 55. The nucleic acid of Example 53 or 54, wherein the nucleic acid further encodes a guide RNA. 56. The nucleic acid of any one of Examples 53 to 55, which is in the form of a vector. 57. The nucleic acid of Example 56, wherein the vector is an expression vector. 58. The nucleic acid of Example 57, wherein the expression vector is a production vector. 59. The nucleic acid of embodiment 57, wherein the expression vector is a delivery vector. 60. The nucleic acid of any one of embodiments 56 to 59, wherein the vector is an RNA vector. 61. The nucleic acid of any one of embodiments 56 to 59, wherein the vector is a DNA vector. 62. The nucleic acid of embodiment 61, wherein the DNA vector is a plasmid. 63. A cell comprising the nucleic acid of any one of embodiments 53 to 62. 64. A cell engineered to express a nucleotide sequence encoding a polypeptide of any one of embodiments 1 to 52. 65. The cell of embodiment 63 or 64, which is a eukaryotic cell. 66. The cell of embodiment 65, which is an insect cell. 67. The cell of Example 65, which is a plant cell. 68. The cell of Example 65, which is a mammalian cell. 69. The cell of Example 68, which is a human cell. 70. A method of producing the polypeptide of any one of Examples 1 to 52, comprising culturing the cell of any one of Examples 63 to 69 under conditions wherein the polypeptide is produced. 71. The method of Example 70, further comprising isolating and/or purifying the polypeptide. 72. A composition comprising: (a) the polypeptide of any one of Examples 1 to 52; and (b) a guide RNA. 73. The composition of Example 72, which is a ribonucleoprotein complex. 74. A composition as in Example 72 or 73, wherein the molar ratio of polypeptide:guide RNA ranges from 1:1 to 1:4. 75. A composition as in Example 72 or 73, wherein the molar ratio of polypeptide:guide RNA ranges from 1:1 to 1:3. 76. A composition as in Example 72 or 73, wherein the molar ratio of polypeptide:guide RNA ranges from 1:1.5 to 1:2.5. 77. A composition as in Example 72 or 73, wherein the molar ratio of polypeptide:guide RNA is 1:2. 78. A method for editing the genome of a cell, comprising introducing into the cell: (a) a polypeptide as in any one of Examples 1 to 52; and (b) a guide RNA. 79. A method of editing the genome of a cell, comprising introducing into the cell one or more nucleic acids encoding: (a) a polypeptide according to any one of embodiments 1 to 52; and (b) a guide RNA, optionally, wherein at least one of the one or more nucleic acids is a nucleic acid according to any one of embodiments 53 to 62. 80. A method of editing the genome of a cell, comprising introducing into the cell: (a) one or more nucleic acids encoding a polypeptide according to any one of embodiments 1 to 52; and (b) a guide RNA. 81. The method according to embodiment 80, comprising contacting the cell with a lipid nanoparticle comprising the one or more nucleic acids and the guide RNA. 82. A method for editing the genome of a cell, comprising introducing into the cell a nucleic acid encoding one or more of: (a) a polypeptide according to any one of embodiments 1 to 52; and (b) a guide RNA, optionally, wherein at least one of the one or more nucleic acids is a nucleic acid according to any one of embodiments 53 to 62. 83. The method according to embodiment 82, comprising contacting the cell with one or more recombinant AAV particles comprising the one or more nucleic acids. 84. The method according to embodiment 82, comprising contacting the cell with one or more lipid nanoparticles comprising the one or more nucleic acids. 85. A method for editing the genome of a cell, comprising introducing into the cell a composition according to any one of embodiments 72 to 77. 86. The method of any one of embodiments 78 to 80, wherein the cell is a mammalian cell. 87. The method of embodiment 86, wherein the mammalian cell is a human cell. 88. The method of embodiment 86 or 87, wherein the mammalian cell is an immune cell, optionally selected from T cells, T cells expressing a chimeric antigen receptor (CAR) or a recombinant TCR, regulatory T cells, bone marrow cells, dendritic cells, and immunosuppressive macrophages. 89. The method of any one of embodiments 78 to 87, wherein the cell is a hematopoietic stem cell, an erythroid progenitor cell, a lymphoid progenitor cell, a peripheral blood mononuclear cell, a T lymphocyte, a B lymphocyte, a macrophage, a monocyte, a neutrophil, an eosinophil, a dendritic cell, or a cell reprogrammed therefrom. 90. The method of any one of embodiments 78 to 89, wherein the cell is a stem cell or a cell differentiated therefrom. 91. The method of embodiment 90, wherein the stem cell is a pluripotent stem cell (PSC) or a cell differentiated therefrom. 92. The method of embodiment 90 or 91, wherein the cell differentiated therefrom is a human immune cell, which is optionally selected from T cells, T cells expressing chimeric antigen receptor (CAR) or recombinant TCR, regulatory T cells, bone marrow cells, dendritic cells and immunosuppressive macrophages. 93. The method of embodiment 90 or 91, wherein the cell differentiated therefrom is a cell of the human nervous system, which is optionally selected from dopaminergic neurons, microglia, oligodendrocytes, astroglial cells, cortical neurons, spinal or oculomotor neurons, intestinal neurons, placode-derived cells, Schwann cells, and trigeminal or sensory neurons. 94. The method of embodiment 90 or 91, wherein the cell differentiated therefrom is a cell of the human cardiovascular system, which is optionally selected from cardiac myocytes, endothelial cells, and nodal cells. 95. The method of embodiment 90 or 91, wherein the cell differentiated therefrom is a cell in the human metabolic system, which is optionally selected from liver cells, bile duct cells and pancreatic β cells. 96. The method of embodiment 90 or 91, wherein the cell differentiated therefrom is a cell in the human eye system, which is optionally selected from retinal pigment epithelial cells, photoreceptor cone cells, photoreceptor rod cells, bipolar cells or ganglion cells. 97. The method of any one of embodiments 78 to 80, wherein the cell is a plant cell. 98. A cell comprising: (a) the composition of any one of embodiments 72 to 77; or (b) the nucleic acid of any one of embodiments 53 to 62. 99. The cell of embodiment 98, which is a mammalian cell. 100. The cell of embodiment 99, wherein the mammalian cell is a human cell. 101. The cell of embodiment 99 or 100, wherein the mammalian cell is an immune cell, which is optionally selected from T cells, T cells expressing chimeric antigen receptors (CAR) or recombinant TCRs, regulatory T cells, bone marrow cells, dendritic cells, and immunosuppressive macrophages. 102. The cell according to any one of embodiments 98 to 100, which is a hematopoietic stem cell, an erythroid progenitor cell, a lymphoid progenitor cell, a peripheral blood mononuclear cell, a T lymphocyte, a B lymphocyte, a macrophage, a monocyte, a neutrophil, an eosinophil, a dendritic cell, or a cell reprogrammed therefrom. 103. The cell according to any one of embodiments 98 to 102, which is a stem cell or a cell differentiated therefrom. 104. The cell according to embodiment 103, wherein the stem cell is a pluripotent stem cell (PSC) or a cell differentiated therefrom. 105. The cell of embodiment 103 or 104, wherein the cell differentiated therefrom is an immune cell, which is optionally selected from T cells, T cells expressing chimeric antigen receptor (CAR) or recombinant TCR, regulatory T cells, bone marrow cells, dendritic cells and immunosuppressive macrophages. 106. The cell of embodiment 103 or 104, wherein the cell differentiated therefrom is a cell of the human nervous system, which is optionally selected from dopaminergic neurons, microglia, oligodendrocytes, astroglial cells, cortical neurons, spinal or oculomotor neurons, intestinal neurons, placode-derived cells, Schwann cells, and trigeminal or sensory neurons. 107. The cell of embodiment 103 or 104, wherein the cell differentiated therefrom is a cell of the human cardiovascular system, which is optionally selected from cardiac myocytes, endothelial cells, and nodal cells. 108. The cell of Example 103 or 104, wherein the cell differentiated therefrom is a cell in the human metabolic system, which is selected from liver cells, bile duct cells and pancreatic β cells as needed. 109. The method of Example 103 or 104, wherein the cell differentiated therefrom is a cell in the human eye system, which is selected from retinal pigment epithelial cells, photoreceptor cone cells, photoreceptor rod cells, bipolar cells or ganglion cells as needed. 110. The cell of Example 98, which is a plant cell. 8. Examples 8.1. Materials and Methods 8.1.1. Structural Modeling of B-GEn.2

The structure of B-GEn.2 has not been characterized to date. The structure of B-GEn.2 was predicted using AlphaFold2 software and compared against a database of proteins with known crystal structures. Based on 14 lead crystal structure hits, a set of candidate structures for B-GEn.2 was generated. This data indicated that the predicted structure of B-GEn.2 was well consistent with several crystal structures from the Bacillusales order of bacteria.

Based on these alignments, NCBI BLASTp searches within the Bacillusales were performed using the OBD II and RuvC I domains of B-GEn.2. Approximately 20 Cas nucleases from related source organisms with approximately 40 to 70% sequence identity were selected for multiple sequence alignment using the MUSCLE alignment algorithm. The spectral relationships of these various nucleases are depicted in the tree of Figure 2, and the alignment itself is shown in Figure 4. 8.1.2. Design of B-GEn.2 variants with amino acid substitutions

Two parallel approaches were used to identify amino acid substitutions that lead to enhanced indel activity of B-GEn.2. In the first approach, DNAStar Lasergene 17 software was used to identify amino acid residues in the AacC2c1 crystal structure that were within 3 angstroms of either the target DNA substrate, guide spacer RNA, or guide tracr RNA. The same procedure was performed on the predicted structure of B-GEn.2, with particular attention paid to those cases where the selected amino acid residues were different from the corresponding residues of AacC2c1.

The second approach involved using Swiss-PdbViewer (also known as Deepview (https://spdbv.unil.ch/)) to identify amino acids within the crystal structure of AacC2c1 (RCSB entry 5U31) that interact via predicted hydrogen bonding with either the target DNA substrate, guide spacer RNA, or guide tracr RNA. Then, by aligning the sequence of AacC2c1 with that of B-GEn.2, a search was performed for instances in which predicted protein-nucleic acid contact residues in AacC2c1 differed on B-GEn.2. To narrow the focus from potentially dozens of candidate mutants to a more manageable number for final protein production and purification, further evaluation focused on Arg or Lys residues that formed close contacts with the target or non-target DNA strands in the AacC2c1 crystal but did not correspond to the predicted B-GEn.2 structure. 8.1.3. Performance and purification of B-GEn.2 variants

Plasmids were constructed for expression of selected point mutants of B.Gen.2. All constructs were expressed in BL21(DE3) cells by first chemically transforming the coding plasmids into these E. coli cells and plating on antibiotic selection LB plates. Colonies were scraped and transferred to 250 mL MagicMedia (Thermo Scientific). Cultures were grown at 37°C for 4 hours and then switched to 16°C for 40 hours for protein expression. Cell pellets were then harvested by centrifugation at 5000 x g for 15 minutes at 4°C and then frozen for later processing. To thaw, the pellet was resuspended in lysis buffer (500 mM NaCl, 50 mM Tris (pH 8.0), 5% glycerol, 5 mM EDTA, 0.5 mM TCEP), sonicated, and the resulting lysate was cleared by centrifugation at 50,000 x g for 30 min at 4°C. The cleared lysate was loaded onto a Heparin Agarose Gel 6 FF column with loading buffer (100 mM NaCl, 50 mM Tris (pH 8.0), 5% glycerol, 5 mM EDTA, 0.5 mM TCEP), followed by a wash step with the same buffer. The protein was eluted using steps of 500 mM, 700, and 1000 mM NaCl using the same buffer. The protein in the main fraction was concentrated to approximately 20 mg/mL. The purity of the protein was estimated by band densitometry of Coomassie-stained SDS-PAGE gels, and aliquots of the purified protein were frozen at -80°C for subsequent use. 8.1.4. RNP production

Purified and concentrated variant B-GEn.2 nuclease was formed into RNPs with a guide RNA of the following sequence targeting a site within the B2M gene, with the spacer underlined: *mU*mA*GCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGACCUAACACGUCUCCAGUCACAACGGCUAAAAAUAGCCAGCAC AGUGUAGUACAAGAGAUAGA*mA*mA*mG (SEQ ID NO: 178)

RNPs were assembled by mixing the B2M targeting guide RNA and nuclease at a molar ratio of 2:1 in a buffer containing 225 mM NaCl and incubated at room temperature for approximately 30 minutes. The complexation efficiency of the RNPs was assessed by cation exchange UPLC using a linear elution gradient of NaCl on an Agilent Bio SCX (NP1.7, SS) column. Buffer A consisted of 10 mM sodium phosphate, 100 mM NaCl, pH 6.5, and buffer B was 10 mM sodium phosphate, 1 M NaCl, pH 6.5. The percent efficiency of complexation was obtained by dividing the peak area of the RNP by the peak area of the total nuclease added (equivalent sample without guide RNA) and multiplying by 100. Values range from 70 to 90% complexation, depending on the nuclease variant tested. The ability of the RNPs to cleave DNA in vitro was assessed using an in vitro plastid cleavage assay. Increasing amounts of RNPs were mixed in CutSmart buffer (New England Biolabs) with a fixed amount of plastids containing the B2M target site that had been linearized using the Xho-I enzyme. After incubation at 37°C for 30 min, the reaction was quenched in the presence of proteinase K at 50°C for 10 min. The samples were applied to D5000 tape and analyzed on an Agilent 4200 TapeStation instrument. The percentage of cleavage for each reaction was calculated as the peak area of the product relative to the peak area of the total substrate added (equivalent sample without RNP) and multiplied by 100. The _EC50 values for each variant protein (or Cpf1 control) series were determined using Prism software from GraphPad (version 9.3.1) (ranging from 0.07 to 0.21 nM). 8.1.5. In-cell gene editing

iPSCs were cultured in substrate-coated T75 flasks with Essential 8 (E8) growth medium and maintained at 37°C and 5% _CO2 between passages. Passaging was completed at 75 to 80% confluency. On the day of nucleofection, iPSCs were detached from the flask using Accutase™ Cell Dissociation Solution (Stem Cell Technologies) by incubation at 37°C for 10 minutes and then quenching with an equal volume of E8 medium. Cell pellets were harvested by centrifugation at 115xg for 3 minutes followed by resuspension in Lonza P3 Primary Cell Nucleofection Buffer. Ribonucleoproteins (RNPs) and sgRNAs were assembled using a 1:2 ratio of protein:sgRNA (IDT) for each nuclease construct. Nucleofection of complexed RNPs into P3 resuspended iPSCs was achieved using the LONZA 4D nucleofection instrument. Nucleofected cells were then seeded at 150,000 cells per well in substrate-coated 24-well Falcon flat-bottom plates (Corning) and grown for 72 to 96 hours in E8 growth medium with Rock inhibitor Y-27632 (Tocris). After harvest, some of the cells were stained for flow cytometry using an anti-B2M APC-binding antibody from BioLegend (used in B2M targeting experiments). The remaining cells were resuspended in 30 µL lysis buffer from BioRad's singleshot cell lysis kit and crude gDNA was extracted by incubation at room temperature for 10 minutes, then at 37°C for 5 minutes, followed by proteinase K inactivation at 75°C for 5 minutes. 1 µL of crude gDNA extract was used directly in each 25 µL PCR reaction for the first step of amplicon sequencing, followed by end-prep and indexing and sequencing on an Illumina MiSeq sequencer. 8.2. Example 1: Design and performance of variant B-GEn.2 with amino acid substitutions

The V-type endonuclease B-GEn.2 (SEQ ID NO: 3) from Brevibacterium species was previously evaluated for gene editing activity in several proprietary iPSC lines. To improve indel formation, a plausible structure of wild-type B-GEn.2 was identified by computer simulation as described in Section 8.1.1. Variant B-GEn.2 sequences with single amino acid site mutations were designed and generated as described in Section 8.1.2. Selected B-GEn.2 variants were expressed in BL21 (DE3) cells and purified as described in Section 8.1.3.

The lead plausible structure of B-GEn.2 was identified to be completely consistent with the previously published crystallographic structures of the Cas nuclease AacC2c1 from Alicyclobacillus acidoterrestris (Figures 3A to 3C) and the Cas nuclease BthC2C1 from the organism Geobacillus thermoleovorans (not shown).

Next, the amino acid sequences of AacC2c1 and B-GEn.2 were aligned to determine amino acid differences at key positions. Sequence alignment between these two nucleases revealed approximately 37% amino acid sequence identity. Using the differences in the amino acid sequences of AacC2c1 and B-GEn.2, non-Arg and non-Lys residues in the predicted B-GEn.2 structure that corresponded to Arg or Lys residues that formed close contacts with target and non-target DNA in the AacC2c1 crystal structure were identified as mutation targets. An initial list of 19 mutation targets was identified using DNA Star and DeepView, as described in Section 8.1.2, from which 8 mutation targets were selected for further evaluation, and their generation and purification were described in Section 8.1.3.

The expression profiles of all eight B-GEn.2 variants were comparable to that of wild-type B-GEn.2 (Table 7). Representative Coomassie-stained SDS-PAGE images of one of the variants (B-GEn.2 D501R) based on single-step heparin sulfate purification are shown in FIG5 . Table 7 B-GEn.2 Measurement concentration (mg/mL) Estimated purity (%) Adjust concentration (mg/mL) WT 9.0 65% 5.8 Variant 1 6.0 61% 3.7 Variant 2 8.9 74% 6.6 Variant 3 6.4 74% 4.7 Variant 4 10.0 59% 5.9 Variant 5 9.0 76% 6.8 Variant 6 17.0 72% 12.2 Variant 7 12.3 59% 7.3 Variant 8 9.0 65% 5.9 8.3. Example 2: Characterization of RNPs with B-GEn.2 variants

Purified B-GEn.2 variants were formed into RNPs with guide RNAs targeting sites within the B2M gene as described in Section 8.1.4. The efficiency of RNP formation and the efficacy of in vitro cleavage of the target B2M site were assessed.

The results demonstrated that the B-GEn.2 variants were able to efficiently complex with the target guide RNA to form RNPs, wherein the complexation efficiency of the B-Gen.2 variants was found to be in the range of 71 to 92%.

The in vitro activity assay indicated that there were no detectable significant differences between the B-GEn.2 variants and WT B-GEn.2. The target plastid cleavage efficiency (as assessed by EC ₅₀ ) of the B-GEn.2 variants was found to be in the range of 0.07 to 0.17 nM. Figure 6 shows the cleavage efficiency of one of the B-GEn.2 variants, B-GEn.2 D501R, relative to the cleavage efficiency of WT B-Gen.2 and Cpf1 at different nuclease: linearized plastid ratios. 8.4. Example 3: Efficient intracellular gene editing of the B2M locus in iPSCs with B-GEn.2 D501R

As described in Section 8.1.4, intracellular gene editing of B-GEn.2 D501R was assessed by examining indel production at the B2M target locus in iPSCs, which was compared to gene editing achieved by WT B-GEn.2 and AsCpf1 Ultra (IDT).

Greater than 80% indel formation efficiency was achieved with B-GEn.2 D501R, which was greater than that achieved by Cpf1 (Figure 7). This indel formation efficiency achieved by B-GEn.2 D501R was 2-3 times higher than that achieved by WT B-Gen.2 (Figure 7). This result suggests that substitution of Asp by Arg at D501, which in the crystal structure of AacC2c1 is shown to hydrogen bond to the +1 phosphate backbone group of the target DNA strand, enhances the ability of the B-GEn.2 variant to cleave its target substrate. 9. Sequence Listing

Exemplary sequences of the present invention are provided in Table 8 below (wherein "SEQ" refers to SEQ ID NO). Table 8 SEQ describe sequence 1 WT B-GEn.1 aa sequence 2 WT B-GEn.1.2 aa sequence 3 WT B-GEn.2 aa sequence 4 B-GEn.1 D504R aa sequence 5 B-GEn.1.2 D501R aa sequence 6 B-GEn.2 D501R aa sequence 7 B-GEn common I aa sequence 8 B-GEn.1 RuvC I domain aa sequence 9 B-GEn.1 RuvC II domain aa sequence 10 B-GEn.1 RuvC III domain aa sequence ADINAAQNLQKRFWL 11 B-GEn.1.2 and B-GEn.2 RuvC I domain aa sequences KELSVLMENTQIGNENGVSTIEAGMRIMSIDLGQRTAAAVSIFEVISKKPDEKETKLFYPIADTDLYAVHRRSLLLRLPGEEISS 12 B-GEn.1.2 and B-GEn.2 RuvC II domain aa sequences CQVILFEDLSRYRFALDRPRRENNRLMKWAHRSIPRLTYMQAELFGIQVGDV 13 B-GEn.1.2 and B-GEn.2 RuvC III domain aa sequences ADINAAQNLQKRFWQ 14 Wild-type SV40 large T protein PKKKRKV 15 Nucleoplasm KRPAATKKAGQAKKKK 16 c-myc PAAKRVKLD 17 EGL-13 MSRRRKANPTKLSENAKKLAKEVEN 18 TUS KLKIKRPVK 19 Alkaline PY-NLS (KR)-X(0,2)-(KR)-(KR)-x(3,10)-(RHK)-X(1,5)-PY 20 SV40 large T protein, variant 1 PKKKRMV twenty one SV40 large T protein, variant 2 PKKKRKWEDP twenty two SV40 large T protein, variant 3 CGYGPKKKRKVGG twenty three SV40 large T protein, variant 4 CGYGPKKKRKV twenty four SV40 large T protein long NLS CYDDEATADSQHSTPPKKKRKWEDPKDFESELLS 25 SV40 large T protein, variant 5 CGGPKKKRKWG 26 SV40 large T protein, variant 6 PKKKIKW 27 SV40 large T protein, variant 7 KRTADGSEFESPKKKRKV 28 Nucleoplasmic min-NLS TKKAGQAKKK 29 Nucleocytoplasmic NLS variant 1 TKKAGQAKKKKLD 30 Nucleocytoplasmic NLS variant 2 CGQAKKKKLD 31 c-myc NLS2 RQRRNELKRSP 32 Polyoma large T protein PKKARED 33 Polyomavirus large T protein CGYGWSRKRPRPG 34 SV40 VP1 capsid polypeptide APTKRKGS 35 Polyomavirus major capsid protein VP1 (11 N-terminal aa) APKRKSGVSKC 36 SV40 VP2 capsid protein (39 kD) PNKKKR 37 Polyomavirus capsid protein VP2 EEDGPQKKKRRL 38 Yeast Histone H2B GKKRSKA 39 Adenovirus E1a KRPRP 40 Adenovirus type 2/5 E1a CGGLSSKRPRP 41 Xenopus N1, NLS1 LVRKKRKTE3SP 42 Xenopus N1, NLS2 LKDKDAKKSKQE 43 V-Rel GNKAKRQRST 44 NS1 protein of influenza A virus PFLDRLRRDQK 45 Human laminin A SVTKKRKLE 46 Xenopus laminin A SASKRRRLE 47 Human c-myc ACIDKRVKLD 48 Mouse c-abl SALIKKKKKMAP 49 Adenovirus 5 DBP PPKKRMRRRIE 50 Rat glucocorticoid receptor YRKCLQAGMNLEARKTKKKIKGIQQATA 51 Human glucocorticoid receptor CGYGARKTKKKIK 52 Human glucocorticoid receptor NLS variants RKCLQAGMNLEARKTKK 53 Rabbit progesterone receptor RKFKKFNK 54 Human estrogen receptor CGYGIRKDRRGGR 55 Human androgen receptor CGYGARKLKKLGN 56 Chicken Ets1 core NLS GKRKNKPK 57 c-myb PLLKKIKQ 58 N-myc PPQKKIKS 59 p53 PQPKKKP 60 p53 NLS variants PQPKKKPL 61 c-erb-A SKRWAKRKL 62 Yeast ribosomal protein L29 NLS MTGSKTRKHRGSGA 63 Yeast ribosomal protein L29 NLS, variant 1 MTGSKHRKHPGSGA 64 Yeast ribosomal protein L29 NLS, variant 2 R 65 Yeast ribosomal protein L29 NLS, variant 3 KRW 66 Yeast ribosomal protein L29 NLS, variant 4 KYRKHP 67 Yeast ribosomal protein L29 NLS, variant 5 KHRR 68 Yeast ribosomal protein L29 NLS, variant 6 KHKKHP 69 Yeast ribosomal protein L29 NLS, variant 7 RHLK 70 Yeast ribosomal protein L29 NLS, variant 8 KHRKYP 71 Yeast ribosomal protein L29 NLS, variant 9 QUR 72 Xenopus N1 NLS1 LVRKKRKTE3SP 73 Xenopus N1 NLS2 LKDKDAKKSKQE 74 VirusJun ASKSRKRKL 75 Human T-cell leukemia virus Tax transactivator protein GGLCSARLHRHALLAT 76 Mouse nuclear MX1 protein (72 kD) DTREKKKFLKRRLLRLDE 77 Mouse nuclear MX1 protein NLS variant REKKKFLKRR 78 Human retinoic acid receptor CGYGDRNKKKKE 79 Human XPAC RKRQRALMLRQAR 80 VirD2 endonuclease for T-DNA ligation EYLSRKGKLEL 206 Putative core NLS of yeast TRM1 KKSKKKRC 81 Human foamy retrovirus Gag protein QPQRYGGGRGRRW 82 SV40 Vp3 structural protein NKKKRKLSRGSSQKTKGTSASAKARH KRRNRSSRS 83 Simian sarcoma virus v-sis gene product RVTIRTWRWRRPPKGKHRK 84 Putative double NLS of Xenopus laevis protein factor Xnf7 KRKIEEPEPEPKKAK 85 Yeast SWI5 gene product KKYENVVIKRSPRKRGRPRKD 86 Herpes simplex virus ICP8 protein GRKRAFHGDDPFGEGPPDKKGD 87 VirD2 endonuclease double NLS KRPREDDDGEPSERKRARDDR 88 IBB domain from importin-α RMRIZFKNKGKDTAELRRRRVEVSVEL RKAKKDEQILKRRNV 89 hRNPAl M9 NLS NQSSNFGPMKGGNFGGRSSGPYGGG GQYFAKPRNQGGY 90 Human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK 91 Hepatitis virus delta antigen RKLKKKIKKL 92 Influenza virus NLS PKKKRK 93 Mouse c-abl IV SALIKKKKKMAP 94 Myoma T protein NLS1 VSRKRPRP 95 Myoma T protein NLS2 PPKKARED 96 myc proto-oncogene protein [Homo sapiens] GPAAKRVKLD 97 Heat shock factor protein HSF8 [Tomato] KKRRIK 98 2x SV40, LrgT PKKKRKVEDPKKKRKVD 99 3x SV40, LrgT PKKKRKVDPKKKRKVDPKKKRKV 100 Single NLS Common Collection 1 (Dissertation Tatyana Goldberg 2016) KKGKKKGK 101 Single NLS Common Collection 2 (Dissertation Tatyana Goldberg 2016) PKRRRGVVL 102 Single NLS Common Collection 3 (Dissertation Tatyana Goldberg 2016) EQLFKRRNV 103 Single NLS Common Collection 4 (Dissertation Tatyana Goldberg 2016) KRRRR 104 Single NLS Common Collection 5 (Dissertation Tatyana Goldberg 2016) KKRR 105 Single NLS Common Collection 6 (Dissertation Tatyana Goldberg 2016) EGAPPAKRPR 106 Single NLS Common Collection 7 (Dissertation Tatyana Goldberg 2016) MLRRRRRKRAR 107 Single NLS Common Collection 8 (Dissertation Tatyana Goldberg 2016) RRKRR 108 Single NLS Common Collection 9 (Dissertation Tatyana Goldberg 2016) RKR 109 Single-linked NLS minor cluster (Dissertation Tatyana Goldberg 2016) FKAVLEDILGEL 110 NLSdb, protein source P10152 KNRRL 111 NLSdb, protein source Q09353 R 112 NLSdb, protein source Q0VD86, Q58DS6, Q5R6G2, Q9ERI5, Q6AYK2, Q6NYC1 RRKKRR 113 NLSdb, protein sources Q99PU7, D3ZHS6, Q92560, A2VDM8 RRKRSR 114 NLSdb, protein source Q14781, P30658 KRKP 115 NLSdb, protein sources P02545, P48678, P48679, Q3ZD69 KKRKLE 116 NLSdb, protein source O35914, Q01954 PKKKSRK 117 NLSdb, protein source Q9FYS5, Q43386 PKRGRGR 118 NLSdb, protein source E5RQA1 KEKRKKR 119 NLSdb, protein source Q9Z1J1, Q9HCS4, Q924A0 KKKKRKR 120 NLSdb, protein source Q80WE1, Q5R9B4, Q06787, P35922 RRGDGRRR 121 NLSdb, protein source Q9Y261, P32182, P35583 LSPSLSPL 122 NLSdb, protein source P07156 VNFSEFSK 123 NLSdb, protein source Q96EB6 IVINILSE 124 NLSdb, protein source Q6AZ28, O75928, Q8C5D8 PPAKRKCIF 125 NLSdb, SeqNLS prediction QRPGPYDRP 126 NLSdb, protein source Q8L7L5, A1L4X7, O80834, Q8LPN5 KRKRGRPRK 127 NLSdb, protein source O88907, O75925 KIKELYRR 128 NLSdb, SeqNLS prediction MVQLRPRASR 129 NLSdb, protein source Q5VK71 KKRREKQRRR 130 NLSdb, protein source P0C6L6, P25880, Q81835, P29833, P25882, P06934, P0C6L3, P29997, P0C6M5, P0C6M9, P29996, P0C6M1, P0C6L8, P0C6M2, P0C6L7, P25881 EGAPPAKRAR 131 NLSdb, protein source Q45FA5 PKKGDKYDKTD 132 NLSdb, protein source P97376, Q14331 KKKKSKDKKRK 133 NLSdb, protein source P21827 AHRAKKMSKTHA 134 NLSdb, protein source Q6ZN17 KKGPSVQKRKKT 135 NLSdb, protein source Q4R8Y1 KGVKRKADTTTP 136 NLSdb, protein source Q91Y44, D4A7T3 KGVKRRADTTTP 137 NLSdb, protein source Q15397, Q8BKS9, Q562C7 KKPKWDDFKKKKK 138 NLSdb, protein source Q96GM8 KRRRRRRREKRKR 139 NLSdb, protein source P61635, Q6DV79, Q19S50, P52631 DVRKRVQDLEQKM 140 NLSdb, protein source O60716 KKGKDEWFSRGKK 141 NLSdb, protein source P30999 KKGKDEWFSRGKKP 142 NLSdb, SeqNLS prediction ASPEYVNLPINGNG 143 NLSdb, protein source Q7Z7C8, Q5ZMS1, Q9EQH4, A7MAZ4 YLRPVKKPKIRRKK 144 NLSdb, protein source O15381 KRKGKLKNKGSKRKK 145 NLSdb, SeqNLS prediction RRRGKNKVAAQNCRK 146 NLSdb, protein source O15516, Q5RAK8, Q91YB2, Q91YB0, Q8QGQ6, O08785, Q9WVS9, Q6YGZ4 DKAKRVSRNKSEKKRR 147 NLSdb, protein source G5EFF5 EEQLRRRKNSRLNNTG 148 NLSdb, protein source P10103, Q4R844, P12682, B0CM99, A9RA84, Q6YKA4, P09429, P63159, Q08IE6, P63158, Q9YH06, B1MTB0 HKKKHPDASVNFSEFSK 149 NLSdb, protein source Q9Z301, O54943, Q8K3T2 KKTGKNRKLKSKRVKTR 150 NLSdb, protein sources Q38740, Q38741, Q700W2, Q9S7A9, Q6Z461, P93015, Q94JW8, Q9S758 KRSCRRRLAGHNERRRK 151 NLSdb, protein source Q501B2 KRQRRKQSNRESARRSR 152 NLSdb, SeqNLS prediction RGKGGKGLGKGGAKRHRK 153 NLSdb, protein source Q63014, Q9DBR0 RRRGFERFGPDNMGRKRK 154 NLSdb, protein source Q0IJ08, Q2TAE3, Q63470, Q13627, Q61214 RRHQQGQGDDSSHKKERK 155 NLSdb, protein source Q8LAM0, O24454 KKKTGVIAPKRFVQRLKK 156 NLSdb, protein source Q0E671 KRAMKDDSHGNSTSPKRRK 157 NLSdb, protein source Q9P127 KVNFLDMSLDDIIIYKELE 158 NLSdb, SeqNLS prediction KKYENVVIKRSPRKRGRPRK 159 NLSdb, SeqNLS prediction KRGNSSIGPNDLSKRKQRKK 160 NLSdb, protein source Q9BZZ5, Q5R644 KRASEDTTSGSPPKKSSAGPKR 161 NLSdb, SeqNLS prediction KRIHSVSLSQSQIDPSKKVKRAK 162 NLSdb, SeqNLS prediction EVLKVIRTGKRKKKAWKRMVTKVC 163 NLSdb, SeqNLS prediction IINGRKLKLKKSRRRSSQTSNNSFTSRRS 164 NLSdb, protein source Q76IQ7 AHFKISGEKRPSTDPGKKAKNPKKKKKKDP 165 Flexible connector (GGGGS) ₃ 166 Hybrid connector GGG(EAAAK) ₃ 167 Rigid connector A(EAAAK) ₄ ALE(EAAAK) ₄ A 168 Flexible connector 2 GGGGSLVPRGSGGGGS 169 Proline-rich, rigid, interdomain linker GAAPAAAPAKQEAAAAPAPAAKAAEAPAAAPAAKA 170 Cuttable connector THRQPRGWE 171 B-GEn.2-sgRNA_v4 172 B-GEn.2-sgRNA_v4.2 173 B-GEn.2-sgRNA_v4.3 174 B-GEn.2-sgRNA_v4.4 175 B-GEn.2-sgRNA_v4.5 176 Tracr B-GEn.2 177 Tracr B-GEn.1 178 Guide RNA targeting B2M 179 BkaCas12b aa sequence 180 BcyCas12b aa sequence 181 BacCas12b aa sequence 182 BmaCas12b aa sequence 183 BshCas12b aa sequence 184 BagCas12b aa sequence 185 AacC2c1 aa sequence 186 BsyCas12b aa sequence 187 Bv3Cas12b aa sequence 188 PteCas12b aa sequence 189 BcoCas12b aa sequence 190 LseCas12b aa sequence 191 BheCas12b aa sequence 192 BmeCas12b aa sequence 193 BcaCas12b aa sequence 194 BpaCas12b aa sequence 195 BpuCas12b aa sequence 196 BhiCas12b aa sequence 197 BgeCas12b aa sequence 198 BkaCas12b aa sequence 199 BthCas12b aa sequence 200 B-GEn.2(D501R)-NLS aa sequence 201 B-GEn.1 (D504R) target interaction strand aa sequence GPAFLNVVLRL 202 B-GEn.1.2 (D501R) target interaction strand aa sequence GPIFLNVVVRV 203 B-GEn.2 (D501R) target interaction strand aa sequence GPIFLNVVVRV 204 Common target interaction strand aa sequence _{GX1X2X3X4NX5X6X7DX8X1 :} any amino _acid ; in some embodiments, _X1 is D, E, S, P, K, or _{R. X2 : any} amino _acid ; in some embodiments, _X2 is V, I, or _{A. X3} : any amino acid; in some embodiments, _X3 is Y or F. _X4 : any amino acid; in some embodiments, _X4 is L or F. X5: any amino acid; in some embodiments, _X5 is I, L, F, V, or _{M. X6} : any amino acid; in some embodiments, _X6 is S, V, T, or A. _X7 : any amino acid; in some embodiments, _X7 is V, L, or I. _X8 : any amino acid; in some embodiments, _X8 is V, F, L, or I. 205 B-GEn common II aa sequence 207 Common aa sequences of Cas nucleases in Bacillusales 10. Incorporation by Reference

All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. If there is any inconsistency between the teachings of this document and one or more of the references incorporated herein, the teachings of this specification shall prevail.

Figure 1 shows the amino acid sequence alignment of three B-GEn enzymes: B-GEn.1 (SEQ ID NO: 1), B-GEn.1.2 (SEQ ID NO: 2) and B-GEn.2 (SEQ ID NO: 3) and an exemplary common sequence (SEQ ID NO: 205). The residues of each B-GEn enzyme that differ from the corresponding amino acids of the other two B-GEn enzymes are shown in dark boxes, while the matching amino acids are unlabeled.

Figures 2A to 2C show phylogenetic evaluation of endonucleases derived from various species in the order Bacillales. Figure 2A is a phylogenetic tree showing genera derived from the genus Bacillus, adapted from Suzuki, 2018, Appl Microbiol Biotechnol. 102:10425-10437. Figure 2B shows the phylogenetic origins of several endonucleases. Figure 2C is a phylogenetic tree generated using the tree building algorithm of Geneious Prime software based on the NCBI blast output of the B-GEn.2 OBD II domain and RuvC I domain sequences, showing endonucleases derived from the genus Bacillus of the order Bacillales.

Figures 3A to 3C show the AacC2c1 and B-GEn.2 enzyme structures. Figure 3A shows the crystal structure of AacC2c1 (5U33) visualized using DNAStar. Figure 3B shows the predicted structure of B-GEn.2 generated using AlphaFold2 and visualized using DNAStar. Figure 3C shows the alignment of the crystal structure of AacC2c1 (5U33) with the predicted structure of B-GEn.2.

Figure 4 shows the amino acid sequence alignment of B-GEn.1 and B-GEn.2 with 20 different Cas nucleases of the Bacillus order (created using the MUSCLE alignment algorithm of Geneious Prime software). The domains are labeled on the Bth C2C1 sequence as follows: bold amino acids represent oligonucleotide binding domains (OBD domains) (OBD-I and OBD-II), single consecutive underlined amino acids represent recognition (REC) domains (REC1-I, REC1-II, and REC2), italic amino acids represent PAM-interaction (PI) domains, and double consecutive underlined amino acids represent RuvC domains (RuvC-I, RuvC-II, RuvC-III), which together form the nuclease domain. The plus sign above the consensus sequence indicates the amino acid corresponding to the D501 residue of B-GEn.2. The asterisk above the residue marks the active site catalytic residue within the RuvC domain.

FIG. 5 is a Coomassie-stained image of an SDS-PAGE gel showing the results of a single-step heparin sulfate-based purification of B-GEn.2 D501R using the method described in Section 8.1.3.

FIG. 6 is a graph showing the cleavage efficiency of the B2M target site by B-GEn.2 D501R relative to B-GEn.2 WT and Cpf1 in an in vitro plastid cleavage assay at different nuclease: linearized plastid ratios.

Figure 7 is a bar graph showing the results of two evaluations of B2M gene editing in iPSCs using 50 pmol RNPs containing B-GEN.2 WT, B-GEn.2 D501R, or Cpf1. The percentage of indel formation on the Y-axis represents the percentage of gene editing with the RNPs formed by the indicated enzymes.

Figure 8 shows the structure of the superposition of AacC2c1 (labeled 5U31 in the image) and B-GEn.2 generated using AlphaFold2. Both AacC2c1 (black residues) and B-GEn.2 (white residues) adjacent to the P-1 nucleotide of the target DNA show the aligned amino acid backbones of the target interaction strands of the superpositioned endonucleases, which are located near the PAM recognition region of each enzyme and include amino acids corresponding to R507 of AacC2c1 (showing a side chain hydrogen-bonded to a phosphate group in one of the DNAs) and D501 of B-GEn.2 (grey, away from the target DNA strand). The amino acid sequence of the target interaction strand of B-GEn.1 with a D504R substitution corresponds to SEQ ID NO: 201, the amino acid sequence of the target interaction strand of B-GEn.1.2 with a D501R substitution corresponds to SEQ ID NO: 202, and the amino acid sequence of the target interaction strand of B-GEn.2 with a D501R substitution corresponds to SEQ ID NO: 203. The common amino acid sequence of the target interaction strand among the endonucleases illustrated in FIG. 4 (and wherein the amino acid arginine is at a position corresponding to D501 of SEQ ID NO: 3) is provided as SEQ ID NO: 204.

TW202521691A_113137996_SEQL.xml

Claims (33)

An engineered V-type endonuclease comprising an amino acid other than aspartic acid at a position corresponding to D504 of SEQ ID NO: 1 (B-GEn. 1) or D501 of SEQ ID NO: 2 (B-GEn. 1.2) or SEQ ID NO: 3 (B-GEn. 2). The engineered V-type endonuclease of claim 1, which is an engineered B-GEn polypeptide. The engineered V-type nuclease of claim 1 or claim 2, which has arginine at the position corresponding to D504 of SEQ ID NO: 1 (B-GEn.1) or D501 of SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2). An engineered V-type nuclease, which is optionally an engineered V-type nuclease as in any one of claims 1 to 3, comprising the following amino acid sequence: (a)    having a substitution at a position corresponding to D501 of SEQ ID NO: 3, wherein the substitution: (i) is a substitution D501R compared to the amino acid sequence of SEQ ID NO: 3; or (ii)     increases gene editing efficiency activity compared to the corresponding amino acid sequence without the substitution at position D501; and (b)    has at least 90% sequence identity with SEQ ID NO: 1 (B-GEn.1), SEQ ID NO: 2 (B-GEn.1.2) or SEQ ID NO: 3 (B-GEn.2). An engineered V-type endonuclease comprising the amino acid sequence of SEQ ID NO: 4. An engineered V-type endonuclease comprising the amino acid sequence of SEQ ID NO: 5. An engineered V-type endonuclease comprising the amino acid sequence of SEQ ID NO: 6. An engineered V-type endonuclease as claimed in any one of claims 1 to 7, comprising at least one NLS, optionally located at the C-terminus of the amino acid sequence, and optionally wherein: (a)    the engineered V-type endonuclease lacks any NLS located at the N-terminus of the amino acid sequence; or (b)    the engineered V-type endonuclease comprises at least one NLS located at the N-terminus of the amino acid sequence. A nucleic acid comprising a nucleotide sequence encoding the engineered V-type endonuclease of any one of claims 1 to 8. The nucleic acid of claim 9, wherein the nucleotide sequence encoding the engineered V-type endonuclease of any one of claims 1 to 8 can be operably linked to a promoter. The nucleic acid of claim 9 or claim 10, wherein the nucleic acid further encodes a guide RNA. The nucleic acid of any one of claims 9 to 11, which is in the form of a vector. The nucleic acid of claim 12, wherein the vector is a recombinant adeno-associated virus (AAV) vector. A cell comprising the nucleic acid of any one of claims 9 to 13. A cell engineered to express a nucleotide sequence encoding the engineered V-type endonuclease of any one of claims 1 to 8. The cell of claim 14 or claim 15, which is a eukaryotic cell, and optionally, the eukaryotic cell is a human cell. A method for producing an engineered V-type nuclease as in any one of claims 1 to 8, the method comprising culturing a cell as in any one of claims 14 to 16 under conditions in which the engineered V-type nuclease is produced, the method further comprising isolating and/or purifying the engineered V-type nuclease as desired. A composition comprising: (a)    an engineered V-type nuclease as in any one of claims 1 to 8; and (b)    a guide RNA. The composition of claim 18, which is a ribonucleoprotein complex. A composition as claimed in claim 18 or claim 19, wherein the molar ratio of the engineered V-type nuclease:guide RNA ranges from 1:1 to 1:4. A method for editing the genome of a cell, the method comprising introducing into the cell: (a)    an engineered V-type nuclease as in any one of claims 1 to 8; and (b)    a guide RNA. A method for editing the genome of a cell, the method comprising introducing into the cell: (a)    one or more nucleic acids encoding an engineered V-type endonuclease as described in any one of claims 1 to 8; and (b)    guide RNA. The method of claim 22, comprising contacting the cell with a lipid nanoparticle comprising the one or more nucleic acids and the guide RNA. A method for editing the genome of a cell, the method comprising introducing into the cell a nucleic acid encoding one or more of the following: (a)    an engineered V-type endonuclease as in any one of claims 1 to 8; and (b)    a guide RNA, optionally, wherein at least one of the one or more nucleic acids is a nucleic acid as in any one of claims 9 to 13. The method of claim 24, comprising contacting the cell with one or more recombinant AAV particles comprising the one or more nucleic acids. The method of claim 24, comprising contacting the cell with one or more lipid nanoparticles comprising the one or more nucleic acids. A method for editing the genome of a cell, the method comprising introducing the composition of any one of claims 18 to 20 into the cell. The method of claim 24, wherein the composition is a ribonucleoprotein complex. The method of claim 26 or claim 27, wherein the composition is introduced into the cell via lipid nanoparticles. The method of any one of claims 21 to 29, wherein the cell is a mammalian cell, optionally a human cell. The method of claim 30, wherein the cell is a stem cell or a cell differentiated therefrom, and optionally, wherein the stem cell is a pluripotent stem cell (PSC) or a cell differentiated therefrom. A cell comprising: (a)    a composition as described in any one of claims 18 to 20; or (b)    a nucleic acid as described in any one of claims 9 to 13. The cell of claim 32 is a mammalian cell, and optionally a human cell.