KR20260007576A - Modified guide RNA - Google Patents

Abstract

The present invention relates to modified guide RNA molecules, compositions, and methods for site-specific gene editing and genome modification, such as DNA cleavage, gene activation, or gene suppression. The modified guide RNAs have a modified secondary structure (e.g., a long upper stem and a modified hairpin structure), which allows them to specifically target a target DNA sequence and reduce off-target activity.

Description

Modified guide RNA

This application claims priority to U.S. Provisional Application No. 63/462,873, filed April 28, 2023, entitled "Modified Guide RNA," the contents of which are incorporated herein by reference in their entirety.

This application is filed with a sequence listing submitted electronically in XML format. The sequence listing, titled "BEM_020WO1_SL_xml," was created on April 26, 2024, and is 63,829 bytes in size; the information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

Nuclease, Cas9, is guided to a specific DNA sequence, i.e., the target sequence, by a small RNA molecule called a guide RNA (gRNA) for genetic modification. The activity of CRISPR-Cas9 depends on the sequence and structure of the gRNA. Complete guide RNAs include tracrRNA (trRNA) and crisprRNA (crRNA). The crRNA, which contains the guide region, can form a complete gRNA when covalently or non-covalently linked to the trRNA. The trRNA and crRNA can be contained within a single guide RNA (sgRNA) or in two separate RNA molecules.

Guide RNAs, either as a single guide RNA (sgRNA) or as two individual crRNA and tracRNA molecules, form a common secondary structure, particularly the scaffold sequence of the guide RNA. However, the formation of undesired secondary structures within the guide RNA (gRNA) can inhibit the CRISPR-Cas9 system.

Modifications of guide RNA that increase stability and on-target specificity would be useful.

The present application provides, among other things, modified gRNA molecules, compositions, and methods for site-specific gene editing and genome modification, such as DNA cleavage, gene activation, or gene suppression. The modified guide RNAs have modified secondary structures ( e.g., a long upper stem and a modified hairpin structure). The modified gRNAs provided herein reduce off-target activity while retaining the ability to specifically target DNA sequences.

In one aspect, the present invention encompasses the use of modified single guide RNAs (sgRNAs) to enhance genome editing, for example, editing of target nucleic acids within primary cells (e.g., in cultured test tubes for ex vivo therapeutic applications) or within cells of a subject, such as a human. The present invention also provides methods for treating a disease in a subject by enhancing precise genome editing to correct mutations in target genes associated with the disease. The present invention can be used in any cell type and any genetic locus suitable for nuclease-mediated genome editing technology.

In one aspect, the present invention provides a modified guide RNA (gRNA) comprising a long (or extended) upstream stem comprising at least four base pairs formed by complementary nucleotides, wherein one or more or all nucleotides of the upstream stem are chemically modified nucleotides. In some embodiments, the long (or extended) upper stem comprises 4 to 8 base pairs formed by complementary nucleotides, wherein one or more or all nucleotides of the upper stem are modified nucleotides. In one embodiment, all nucleotides of the extended upper stem are chemically modified.

In some embodiments, the modified gRNA comprises a long upstream stem region comprising 5 to 15 base pairs formed by complementary nucleotides. In one embodiment, the long upstream stem region comprises 5 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 6 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 7 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 8 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises nine base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises ten base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises eleven base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises twelve base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises thirteen base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 14 base pairs formed by complementary nucleotides. In one embodiment, the long upstream stem region comprises 15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 15 to 20 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 20 to 200 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 20 to 40, 40 to 80, 80 to 120, 120 to 160, or 160 to 200 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).

In some embodiments, all nucleotides in the upstream stem of a modified gRNA described herein are chemically modified nucleotides. In other embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleotides in the upstream stem of the modified gRNA are chemically modified nucleotides.

In some embodiments, the modified gRNA described herein further comprises one or more modified nucleotides within the hairpin 1 and hairpin 2 regions. In some embodiments, all nucleotides within the hairpin 1 and 2 regions are modified nucleotides. In other embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleotides within the hairpin 1 and 2 regions of the modified gRNA are modified nucleotides.

In some embodiments, the modified gRNA described herein further comprises a modified stable hairpin 1 region, wherein the modified stable hairpin 1 region comprises an extended stem region comprising at least four base pairs, and the loop of the hairpin 1 comprises a locked nucleic acid. In some embodiments, the extended stem region of the modified stable hairpin comprises eight base pairs.

In one embodiment, the present invention provides GUUUUAGA N _xn GAAA A single guide RNA (sgRNA) comprising the sequence Ny _n AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 23) is provided, wherein N _xn and N _yn are complementary nucleotides having the same number of nucleotides and forming base pairs, the nucleotides of N _xn and N _yn are backbone modified nucleotides, and n is an integer from 5 to 15. In some embodiments, GAAA is a GNRA tetraloop used to lock a hairpin structure. Other GNRA tetraloops include, but are not limited to, GUGA, GCAA, GAGA, GUAA, GGGA, GCGA, and GGAA. In some embodiments, another RNA tetraloop UNCG is incorporated into the sgRNA that locks the hairpin structure. Exemplary UNCG tetraloops include, but are not limited to, UUCG, UACG, UCCG, and UGCG. In some embodiments, the 5' and 3' terminal nucleotides of the sgRNA are backbone modified nucleotides. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGA m(N _xn )mGmAmAmA m( Nyn)AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 24), wherein "m" represents a 2'-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise 5 nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x GAAAN _y N _y N _y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 2). In some examples, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmNxmNxmNxmNxmNxmGmAmAmAmNymNymNymNyAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 25), wherein “m” represents a 2’-OMe modification .

In some examples, Nx and Ny are each complementary nucleotides and comprise six nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x GAAAN _y N _y N _y N _{y N y} N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 3). In some examples, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _x mN _{x mN x} mGmAmAmAmN _y mN _y mN _y mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 26), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise 7 nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N _x GAAAN _y N _y N _{y N y} N y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4). In some examples, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _{x mN x mN x} mGmAmAmAmN _y mN _y mN _y mN _y mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 27), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise eight nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N _x N _x GAAAN _{y N y} N _y N _{y N y} N _y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 5). In some examples, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _{x mN x} mN _{x mN x} mGmAmAmAmN _y mN _y mN _y mN _y mN _y mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmCmGmGmUmGmCUmUmUmU (SEQ ID NO: 28), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise nine nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N _x N _x N x _{N x} GAAAN _{y N y N y N y} N _y N y _{N y} N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 6). In some examples, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _{x mN x mN x mN x mN x mGmAmAmAmN y mN y mN y mN y} mN _y mN _y mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 29), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise 10 nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N _x N _x N _x N _{x N x} GAAAN _y N _y N _y N _y N y N y N _{y N y N y} N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 7). In some examples, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _{x mN x mN x mN x mN x} mN _x mN _{x mGmAmAmAmN y mN y} mN _y mN _y mN _y mN _y mN _y mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 30), wherein “m” represents a 2’-OMe modification.

In one embodiment, the invention provides an sgRNA comprising the sequence GUUUUAGAGCGCGGAAACGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 8). In one embodiment, the invention provides an sgRNA comprising the sequence GUUUUAGAmGmCmGmCmGmGmAmAmAmCmGmCmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 31), wherein "m" represents a 2'-OMe modification.

In another embodiment, the sgRNA of the present invention comprises the sequence of GUUUUAGAGCCGGCGGAAACGCCGGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 9). In another embodiment, the sgRNA of the present invention comprises the sequence of GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmGmUmCmGmUmGmCmUmUmU (SEQ ID NO: 32), wherein "m" represents a 2'-OMe modification.

In some embodiments, one or more nucleotides within the hairpin 1 and hairpin 2 regions of an sgRNA described herein are modified nucleotides. In other embodiments, all nucleotides within the hairpin 1 and hairpin 2 regions of an sgRNA described herein are modified nucleotides. In some embodiments, the loop of hairpin 1 of an sgRNA comprises a 2'-O-methyl modified nucleotide (e.g., a 2'-O-methyl 3'-phosphorothioate (MS) nucleotide, a 2'-O-methyl 3'-thioPACE (MSP) nucleotide), a 2'-F modified nucleotide, a locked nucleic acid, MOE (methoxyethyl), a DNA nucleotide functionalized for conjugation, and combinations thereof.

In some embodiments, the present invention provides a single guide RNA (sgRNA) comprising the sequence GUUUUAGAN _xn GAAAN _yn AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 10), wherein N _xn and N _yn are complementary nucleotides having the same number of nucleotides and forming base pairs, the nucleotides of N _xn and N _yn are modified nucleotides, and n is an integer from 5 to 15, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the N _xn and N _yn nucleotides are 2'-O-methyl modified nucleotides. In one example, the sgRNA comprises the sequence GUUUUAGAm(N _xn )mGmAmAmAm(Nyn)AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 33), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise 5 nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x GAAAN _y N _y N _y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 11). In some embodiments, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _x mN _x mGmAmAmN _{y mN y} mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 34), wherein “m” represents a 2’-O-methyl modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise six nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x GAAAN _y N _y N _y N _y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 12). In some embodiments, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _x mN _{x mN x} mGmAmAmN _y mN _y mN _y mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 35), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise 7 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N _x GAAAN _{y N y N} y N _y N y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 13). In some embodiments, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _x mN _{x mN x mN x mGmAmAmN y} mN _y mN _y mN _y mN y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 36), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise eight nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N _x N _x GAAAN _{y N y} N _y N y N _y N _y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 14). In some embodiments, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _{x mN x} mN _{x mN x} mGmAmAmN _{y mN y mN y} mN _y mN _y mN y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 37), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise nine nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N x N _x N _{x N x} GAAAN _{y N y N y N y} N _y N y N _y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 15). In some embodiments, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _x mN _{x mN x mN x mN x mN x} mGmAmAmAmN _y mN _y mN _y mN _y mN _y mN _y mN _y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmUmUmUmU (SEQ ID NO: 38), wherein “m” represents a 2’-OMe modification.

In some examples, Nx and Ny are each complementary nucleotides and comprise 10 nucleotides forming an upstream stem region of the sgRNA, and the sgRNA comprises the sequence GUUUUAGAN _x N _x N _x N _x N _x N _x N _x N _x N _x N x _{N x} GAAAN _y N _y N _{y N y} N _y N y N _y N _{y N y} N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 16). In some embodiments, each of the Nx and Ny nucleotides is backbone modified. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmN _x mN _x mN _{x mN x mN x mN x mN x mN x} mN _x mGmAmAmAmN _y mN _y mN _y mN _y mN _y mN _y mN y mN y mN _y AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmmN _y AmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmGmUmGmCmUmUmUmU (SEQ ID NO: 39), wherein “m” represents a 2’-OMe modification.

In one embodiment, the sgRNA described herein comprises the sequence GUUUUAGAGCCGGCGGAAACGCCGGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUUUUU (SEQ ID NO: 17). In some embodiments, the sgRNA comprises a backbone modified nucleotide. As a non-limiting example, the sgRNA comprises the sequence GUUUUAGAmGmCmCmGmGmCmGmGmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmUmUmU (SEQ ID NO: 40) (m = 2'-OMe modification).

In some embodiments, the present invention provides a single guide RNA (sgRNA) comprising the sequence of GUUUUAGAN _xn GAAAN _yn AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACN _z N _z N _z N _z GUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 41), wherein N _xn and N _yn are complementary nucleotides having the same number of nucleotides and forming base pairs, the nucleotides of N _xn and N _yn are modified nucleotides, n is an integer from 5 to 15, and four nucleotides of the loop of hairpin 1 (N _z N _z N _z N _z ) comprise UUCG, CUUG or GCAA.

In some embodiments, the sgRNA comprising a long upper stem and a stable hairpin comprises one or more modified nucleotides within the hairpin 1 and hairpin 2 regions. In other embodiments, the sgRNA comprising a long upper stem and a stable hairpin comprises modified nucleotides within the hairpin 1 and hairpin 2 regions. The modifications include 2'-O-methyl modified nucleotides (e.g., 2'-O-methyl 3'-phosphorothioate (MS) nucleotides, 2'-O-methyl 3'-thioPACE (MSP) nucleotides), 2'-F modified nucleotides, and combinations thereof.

In one example, at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

In some embodiments, the loop of hairpin 1 comprises a locking nucleic acid.

In some embodiments, the gRNA described herein further comprises a spacer sequence at the 5'' end of the sgRNA; wherein the spacer sequence comprises a sequence complementary to a target sequence of interest. In some embodiments, the spacer sequence comprises about 18 to 25, or 18 to 30, or 20 to 25, 20 to 30, 15 to 50, 20 to 50, or 20 to 40 nucleotides. As a non-limiting example, the spacer sequence comprises 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides.

In some implementations, the 3' end of the sgRNA described herein is modified.

In some implementations, the 5' end of the sgRNA described herein is modified.

In some implementations, the 3' and 5' ends of the sgRNA described herein are modified.

In some implementations, at least the first three nucleotides at the 5' end of the sgRNA described herein are modified nucleotides.

In one embodiment, the sgRNA comprises a modification at the 5' end of the sequence and a modification at the 3' end of the sequence.

In some embodiments, about 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 75%, 80%, 90%, or 100% of the nucleotides of an sgRNA described herein are modified nucleotides.

In one embodiment, the gRNA described herein comprises the sequence set forth in SEQ ID NO: 20. In one embodiment, the gRNA described herein comprises the sequence set forth in SEQ ID NO: 21. In one embodiment, the gRNA described herein comprises the sequence set forth in SEQ ID NO: 51. In one embodiment, the gRNA described herein comprises the sequence set forth in SEQ ID NO: 52.

In some embodiments, the sgRNA described herein further comprises a nuclear localization sequence (NLS).

In some embodiments, the sgRNA described herein comprises about 102 to 150 nucleotides, about 102 to 120 nucleotides, or about 100 to 180 nucleotides.

In some embodiments, the sgRNAs of the present invention increase gene modification efficacy by about 2 to 1000-fold, or about 2 to 100-fold, or about 2 to 10-fold. As non-limiting examples, the sgRNAs described herein increase gene modification efficacy by 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 550, 600, 650, 700, 750, 800, 900, or 1000-fold.

In another aspect of the present invention, a genetic modification system comprising (a) a CRISPR-associated protein (Cas) polypeptide, or a variant thereof; and (b) a single guide RNA (sgRNA) as described herein is provided.

In some embodiments, the Cas polypeptide is a Cas9 protein, including but not limited to S. pyogenes Cas9 and S. aureus Cas9.

In some embodiments, the Cas9 polypeptide is nickCas9 or dCas9.

In some embodiments, the composition further comprises a single guide RNA as described herein. The composition may further comprise a Cas9 polypeptide, or a variant thereof. In some examples, the Cas polypeptide is a Cas9 protein, dCas9, or a nickcase.

In some embodiments, the compositions described herein are formulated in lipid nanoparticles.

In another aspect, the present invention provides a method for modifying a target gene in a cell, comprising the step of introducing into the cell a gene editing system comprising a single guide RNA (sgRNA) of the present invention.

In some embodiments, the method comprises introducing into a cell a modified single guide RNA (sgRNA) comprising a first nucleotide sequence complementary to a target nucleic acid and a second nucleotide that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein at least one of the nucleotides of the first nucleotide sequence and/or the second nucleotide sequence is a modified nucleotide; and a recombinant expression vector comprising a Cas polypeptide, an mRNA encoding the Cas polypeptide, and/or a nucleotide sequence encoding the Cas polypeptide.

Figure 1A is The hairpin structures of exemplary terminally modified sgRNAs, standard highly modified sgRNA variants, and the LONGEST gRNA design, which includes 5 base pairs in the upper stem of the gRNA, are shown. The chemically modified (terminally modified and highly modified) sgRNAs only include 4 base pairs in the upper stem.
Figure 1B shows exemplary ALAS1-targeted editing in the liver, demonstrating that editing efficiency decreased when the gRNA hairpin was extended without nucleotide modification. Editing efficiency increased when extended in combination with nucleotide modification. gRNA9 contains an internally unmodified extended upper stem (LONGEST 1) that includes the upper stem of the extended hairpin. sgRNA 10 contains another internally unmodified extended upper stem (LONGEST 2) that includes the upper stem of the extended hairpin. sgRNA3 is LONGEST1 containing a 2'OMe-modified nucleotide (sequence shown in SEQ ID NO: 51). ALAS1 editing was performed using the base editor ABE8.8.
Figure 2A shows the hairpin structure of ALAS1 targeting gRNA with terminal modification (gRNA 1), or standard heavy chain modification (gRNA 2), or LONGEST 1 with nucleotide modification (gRNA 3).
Figure 2B shows the ALAS1 targeting gRNA editing efficacy of lipid 1 at various doses.
Figure 2C shows the ALAS1 targeting gRNA editing efficacy of lipid 2 at various doses.
Figure 3A shows the hairpin structure of a gRNA with a terminal modification (EM), a LONGEST modification, a GOLD modification, or a LONGEST_GOLD combination modification.
Figure 3B shows the in vivo editing efficacy of various gRNA modifications at three different target sites (TSBTx3228, TSBTx3215, and TSBTx3222).
Figure 4A is Shown are the hairpin structures of gRNA 4 (standard terminal variant), gRNA 5 (GOLD variant), and gRNA 6 (LONGEST-GOLD variant).
Figure 4B shows the editing efficacy of gRNA 4, gRNA 5, and gRNA 6 after 5 days.
Figure 5 shows the editing efficacy of three novel hairpin extensions (LONGEST 3, LONGEST 4, and unmodified LONGEST 4) in an ALAS1-targeting gRNA compared to the same gRNA with a standard heavy-mod, terminal-modified, or LONGEST modification.

definition

To facilitate a better understanding of the present invention, certain terms are first defined below. Additional definitions of these and other terms are provided throughout this specification.

The article "singular" is used herein to refer to one or more than one ( i.e. , at least one) of the grammatical object of the article. For example, "an element" means one or more than one element.

The terms "approximately" or "about" as used herein, applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain implementations, the terms "approximately" or "about" refer to a range of values that are within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise specified or clear from context (except where such a value exceeds 100% of the possible values).

When referring to a range of numbers in this specification, each number therebetween is explicitly considered to have the same precision. For example, in the range 6 to 9, the numbers 7 and 8 are considered in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

When used to define products, compositions, and methods, the term "comprising" as used herein is intended to mean that the products, compositions, and methods include the recited ingredients or steps, but not to the exclusion of other ingredients or steps. The terms "comprise," "include," "having," "has," "can," "contain," and variations thereof, as used herein, are intended to be open-ended transition phrases, terms, or words that do not exclude the possibility of additional acts or structures. The term "consisting essentially of" means excluding any essential and significant ingredients or steps. Thus, a composition consisting essentially of the recited ingredients would not exclude trace contaminants and pharmaceutically acceptable carriers. "Consisting of" means excluding more than trace elements of other ingredients or methods.

Administration: As used herein, the term "administration" includes administration to a subject, such as oral administration, topical contact, suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration. Administration may be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, and intracranial administration. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

Binding region: As used herein, the term “binding region” refers to a region within a nuclease target region that is recognized and bound by a nuclease, such as Cas9.

Complementarity: As used herein, the term "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, whether through traditional Watson-Crick or other non-traditional methods. The percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that every consecutive residue in a nucleic acid sequence hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, “substantially complementary” refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

Effective amount: As used herein, the term "effective amount" refers to an amount of an agent (e.g., a Cas nuclease, a modified single guide RNA, etc.) sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's body weight and age, the severity of the disease state, the method of administration, and the like, and can be readily determined by one of skill in the art. The specific amount may vary depending on one or more of the following: the specific agent selected, the target cell type, the location of the target cells within the subject, the dosing regimen to be followed, whether it is used in combination with other agents, the timing of administration, and the physical delivery system used.

Efficiency: The term "efficiency" as used herein refers to editing efficiency, or editing percentage; which is the total number of sequence reads with insertions or deletions of nucleotides into the target region of interest relative to the total number of sequence reads after cleavage by the gene editing system of the present disclosure.

Genome : As used herein, the term "genome" refers to the complete set of genes or genetic material present in a cell or organism. The genome includes the DNA or RNA of RNA viruses. The genome includes genes (coding regions), non-coding DNA, and the genomes of mitochondria and chloroplasts.

Genome Editing : As used herein, the term "genome editing" refers to altering genes. Genome editing may involve correcting or restoring a mutated gene. Genome editing may also involve knocking out a gene, such as a mutated or normal gene. Genome editing can be used to treat diseases or enhance muscle recovery by altering a gene of interest.

Guide RNA or gRNA: As used herein, the terms "guide RNA" and "gRNA" are used interchangeably. The terms "guide RNA,""gRNA,""singlegRNA," and "sgRNA," as used interchangeably herein, refer to a short synthetic RNA comprised of a "scaffold" sequence required for Cas9 binding or Cpf1 binding and a user-defined "spacer" or "targeting sequence" (also referred to herein as a protospacer targeting sequence or segment) that defines the genomic target to be modified. As used herein, a "modified gRNA" refers to a gRNA with additional nucleotides or nucleotide modifications.

Hybridization : As used herein, "hybridization" refers to the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a specific mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding between complementary nucleobases.

crRNA: used in this specification The term "crRNA" refers to an RNA sequence comprising a tracrRNA recognition sequence that is complementary to and recognizes a targeting nucleic acid sequence and that binds or is capable of binding to a tracrRNA. The tracRNA recognition portion of the crRNA can bind to the tracrRNA through hybridization or covalent attachment.

tracRNA: As used herein, the term "tracrRNA" refers to a nucleic acid sequence capable of noncovalently binding to a Cas9 protein and binding to a crRNA sequence via hybridization or covalent attachment. In some embodiments, the tracrRNA and crRNA sequences may form a single guide RNA.

Hairpin: As used herein, the term "hairpin" describes a double strand of nucleic acid that is formed when a nucleic acid strand folds and base pairs with another region of the same strand. A hairpin may form a structure comprising a loop or a U-shape. In some embodiments, a hairpin may be composed of an RNA loop. A hairpin may be formed by two complementary sequences in a single nucleic acid molecule that are joined together by a fold or fold of the molecule. In some embodiments, a hairpin comprises a stem or stem-loop structure. In the context of the modified gRNAs described herein, "hairpin region" refers to hairpin 1 and hairpin 2 from the 5' end to the 3' end of the gRNA. The conserved portion of the gRNA is located between hairpin 1 and hairpin 2 of the gRNA.

Stem Loop : As used herein, the term "stem loop" refers to a secondary structure of nucleotides that form a base-paired "stem" that terminates in a loop of unpaired nucleic acids. A stem can be formed when two identical nucleic acid strands are at least partially complementary in sequence when read in opposite directions.

Loop : As used herein, the term "loop" describes a region of unpaired (i.e., non-complementary) nucleotides that can cap a stem. For example, a "tetraloop" describes a loop of four nucleotides. In some embodiments, the upper stem of a modified gRNA may comprise a tetraloop. In some embodiments, GAAA is a GNRA tetraloop, which is used to lock a hairpin structure. In some embodiments, the tetraloop is ANYA, CUYG, GNRA, UNAC, or UNCG.

Pharmaceutically Acceptable Carrier : The term "pharmaceutically acceptable carrier" refers to a substance that facilitates administration of a preparation (e.g., a Cas nuclease, a modified single guide RNA, etc.) to a cell, organism, or subject. A "pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in a composition or formulation and does not cause significant toxicological side effects in a patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, and coloring agents. Those skilled in the art will recognize that other pharmaceutical carriers are useful in the present invention.

Modified or Modified : The term "modified" or "modified", in the context of a guide RNA, refers to various modifications, for example, 2'-O-methyl modified nucleotides (e.g., 2'-O-methyl 3'-phosphorothioate (MS) nucleotides, 2'-O-methyl 3'-thioPACE (MSP) nucleotides), 2'-F modified nucleotides, locked nucleic acids, MOE (methoxyethyl), DNA nucleotides functionalized for conjugation, and combinations thereof.

Nucleic acid: As used herein, the terms "nucleic acid,""oligonucleotide," or "polynucleotide" refer to at least two nucleotides covalently linked. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe capable of hybridizing to a target sequence under stringent hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions. A nucleic acid may be single-stranded or double-stranded, and may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, RNA, or hybrids, both genomic and cDNA, where the nucleic acids can contain combinations of deoxyribonucleotides and ribonucleotides and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be produced by chemical synthesis or recombinant methods.

On-target region : As used herein, the term "on-target region" refers to the target region or sequence of a genome that a gRNA is intended to target. Ideally, the on-target region has complete homology (100% identity or homology) to the target DNA sequence and no homology elsewhere in the genome.

Off-target region : As used herein, the term "off-target region" refers to a region of the genome that has partial homology or partial identity with the on-target region or target region of a gRNA, but which the gRNA is not intended or designed to target.

Subject : The terms "subject,""patient," and "individual" are used interchangeably herein to include a human or an animal. For example, an animal subject can be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, cow, sheep, pig, or goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, rat, guinea pig, or bird), an animal of veterinary significance, or an animal of economic significance.

Treatment: As used herein, the term "treating" refers to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefits and/or prophylactic benefits. A therapeutic benefit refers to a therapeutically relevant improvement or effect on one or more diseases, conditions, or symptoms being treated. For a prophylactic benefit, the composition may be administered to a subject at risk for developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

Modified guide RNA (gRNA)

Provided herein are modified dual guide RNAs (gRNAs) for use in gene editing methods. According to the present disclosure, the modified gRNAs provided herein comprise a long or extended upper stem structure. The modified guide RNAs provided herein may comprise an extended upper stem and a stable, locked hairpin structure. In some aspects, the modified guide RNAs provided herein are single guide RNAs (sgRNAs). The modified sgRNAs of the present disclosure are more stable and exhibit enhanced efficacy in gene editing compared to unmodified sgRNAs.

Long/Extended Upper Stem

In one aspect, the modified gRNA provided herein is a modified single guide RNA (sgRNA) comprising a long (or extended) upstream stem comprising at least four base pairs formed by complementary nucleotides, wherein one or more nucleotides of the upstream stem are chemically modified. Exemplary modifications include 2'OMe modifications, such as 2'-O-methyl (M) nucleotides, 2'-O-methyl 3'-phosphorothioate (MS) nucleotides, 2'-O-methyl 3'-thioPACE (MSP) nucleotides, or combinations thereof.

In some embodiments, the modified sgRNA described herein comprises a long upstream stem region comprising 5 to 15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 5 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 6 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 7 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 8 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises nine base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises ten base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises eleven base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises twelve base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises thirteen base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 14 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upstream stem region comprises 15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).

In some embodiments, all nucleotides in the upstream stem of a modified sgRNA described herein are chemically modified nucleotides. In other embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the upstream stem of a modified sgRNA are chemically modified nucleotides.

In some embodiments, the sgRNA described herein comprises an upper stem modification, wherein the upper stem modification comprises a modification to any one or more of the nucleotides in the upper stem region.

In some embodiments, the sgRNA comprises an upper stem modification, wherein the upper stem modification comprises a modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in the upper stem region.

In some embodiments, the gRNA comprises an upper stem modification, wherein the upper stem modification comprises a modification of about 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 2 to 4, 2 to 6, 2 to 8, 2 to 10, 2 to 12, 2 to 16, 2 to 18, 2 to 20, 4 to 8, 4 to 10, 6 to 12, 6 to 18, 6 to 20, 8 to 10, 8 to 18, 8 to 20, or 10 to 20 nucleotides in the upper stem region.

In some embodiments, the modified nucleotides within the upstream stem region of the sgRNA comprise the same chemical modification. In other embodiments, the modified nucleotides within the upstream stem region of the sgRNA comprise different chemical modifications.

In some embodiments, the sgRNA comprises an upper stem modification, wherein the upper stem modification comprises a 2'-OMe modified nucleotide. In some embodiments, the upper stem modification comprises a 2'-O-MOE modified nucleotide. In some embodiments, the upper stem modification comprises a 2'-F modified nucleotide. In some embodiments, the upper stem modification comprises a 2'-Ome modified nucleotide, a 2'-O-MOE modified nucleotide, a 2'-F modified nucleotide, and/or a combination thereof.

Stable hairpin

In some embodiments, the sgRNA described herein is further modified to include a highly stable hairpin structure. The modified sgRNA described herein further comprises one or more modified nucleotides within the hairpin 1 and hairpin 2 regions. In some embodiments, all nucleotides within the hairpin 1 and 2 regions are modified nucleotides. In other embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleotides within the hairpin 1 and 2 regions of the modified sgRNA are chemically modified nucleotides. In some embodiments, all nucleotides within hairpin 1 and 2 are chemically modified.

In some embodiments, the modified sgRNA described herein further comprises a modified stable hairpin 1 region, wherein the modified stable hairpin 1 region comprises an extended stem region comprising at least four base pairs, and the loop of the hairpin 1 comprises a locked nucleic acid. In some embodiments, the extended stem region of the modified stable hairpin comprises eight base pairs. In some embodiments, the extended stem region of the modified stable hairpin comprises eight base pairs formed by chemically modified nucleotides. As a non-limiting example, all nucleotides of the hairpin comprise a 2'-O-methyl modification.

Highly stable hairpins of sgRNAs are referred to as "locked" hairpins. Locked hairpins can, for example, incorporate one or more locked nucleic acids to form a locked backbone. The term "locked nucleic acid" as used herein refers to a bicyclic RNA analogue in which the ribose is locked in the C3'-endo conformation by introduction of a 2'-O,4'-C methylene bridge. Preferred LNA monomers and methods for their synthesis are also disclosed in U.S. Patent Nos. 6,043,060 and 6,268,490, PCT Application Nos. WO 01/07455, WO 01/00641, WO 98/39352, WO 00/56746, WO 00/56748, and WO 00/66604, and in the following articles: Morita et al., Bioorg. Med. Chem. Lett. 12(1):73-76, 2002; Hakansson et al., Bioorg. Med. Chem. Lett. 11(7):935-938, 2001; Koshkin et al., J. Org. Chem. 66(25):8504-8512, 2001; Kvaerno et al., J. Org. Chem. 66(16):5498-5503, 2001; Halkansson et al., J. Org. Chem. 65(17):5161-5166, 2000; Kvaerno et al., J. Org. Chem. 65(17):5167-5176, 2000; Pfundheller et al., Nucleosides Nucleotides 18(9):2017-2030, 1999; and Kumar et al., Bioorg. Med. Chem. Lett. 8(16):2219-2222, 1998.

In some examples, the hairpin region comprises a locked nucleic acid or LNA comprising a 2'-O,4'-C-methylene ribonucleoside (structure A), wherein the ribose sugar moiety is in a "locked" conformation. The hairpin region comprises at least one 2',4'-C-bridged 2' deoxyribonucleoside (CDNA, structure B). See, e.g., U.S. Patent No. 6,403,566 and Wang et al. (1999) Bioorganic and Medicinal Chemistry Letters, Vol. 9: 1147-1150, both of which are incorporated herein by reference in their entirety. Locked nucleic acids (LNA™) are a class of high-affinity RNA analogues in which the ribose ring is "locked" in a conformation ideal for Watson-Crick binding. LNA™ oligonucleotides exhibit high thermal stability when hybridized to complementary DNA or RNA strands. Additionally, LNA™ oligonucleotides can be made shorter than conventional DNA or RNA oligonucleotides while still maintaining a high Tm. LNA™ oligonucleotides can be composed of a mixture of LNA™ and DNA or RNA. Incorporating LNA™ into oligonucleotides has been shown to improve the sensitivity and specificity of several hybridization-based technologies, including PCR, microarray, and in situ hybridization.

chemical transformation

In some embodiments, the modified gRNA further comprises one or more chemically modified nucleotides. In some embodiments, such modified nucleosides comprise a modified sugar moiety, a modified nucleobase, or both a modified sugar moiety and a modified nucleobase.

In some embodiments, the modified sgRNA comprises one or more modified nucleosides comprising a modified sugar moiety. Such modified sgRNAs comprising one or more sugar-modified nucleosides may have desirable properties, such as improved nuclease stability or increased binding affinity to a target nucleic acid, compared to guide RNAs lacking such sugar-modified nucleosides. In certain embodiments, the modified sugar moiety is a linear modified sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In certain embodiments, the modified sugar moiety is a sugar substitute. Such a sugar substitute may comprise one or more substitutions corresponding to the substitutions of the substituted sugar moiety.

In certain embodiments, the modified sugar moiety is a linear modified sugar moiety comprising a furanosyl ring with one or more acyclic substituents, including but not limited to substituents at the 2' and/or 5' positions. Examples of suitable 2'-substituents for the linear modified sugar moiety include, but are not limited to, 2'-F, 2'-OCH ₃ ("Ome" or "O-methyl"), and 2'-O(CH ₂ ) ₂ OCH ₃ ("MOE"). In certain embodiments, the 2'-substituent is halo, allyl, amino, azido, SH, CN, OCN, CF ₃ , OCF ₃ , OC ₁ -C ₁₀ alkoxy, OC ₁ -C ₁₀ substituted alkoxy, OC ₁ -C ₁₀ alkyl, OC ₁ -C ₁₀ substituted alkyl, S-alkyl, N(R _m )-alkyl, O-alkenyl, S-alkenyl, N(R _m )-alkenyl, O-alkynyl, S-alkynyl, N(R _m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ ON(R _m )(R _n ), or OCH ₂ C( O)―N(R _m )(R _n ), wherein each R _m and R _n is independently H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ alkyl. Specific embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO ₂ ), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of suitable 5'-substituents for the linearly modified sugar moiety include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, the linear modified sugar comprises one or more non-bridging sugar substituents, for example, a 2'-F-5'-methyl sugar moiety (for additional 2', 5'-bis substituted sugar moieties and nucleosides, see PCT International Application WO 2008/101157).

In certain embodiments, the 2'-substituted nucleoside or 2'-linearly modified nucleoside is F, NH ₂ , N ₃ , OCF ₃ , OCH ₃ , O(CH ₂ ) ₃ NH ₂ , CH ₂ CH CH ₂ , OCH ₂ CH CH ₂ , OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ ON(R _m )(R _n ), O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ , and N-substituted acetamides (OCH ₂ C( O)―N(R _m )(R _n )) comprising a sugar moiety comprising a linear 2'-substituent selected from among, wherein each R _m and R _n is independently H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ alkyl.

In certain embodiments, the 2'-substituted nucleoside or 2'-linearly modified nucleoside is selected from the group consisting of F, OCF ₃ , OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂₀ N(CH ₃ ) ₂ , O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ , and OCH ₂ C( O)―N(H)CH ₃ (“NMA”) comprises a sugar moiety comprising a linear 2'-substituent selected from

In certain embodiments, the 2'-substituted nucleoside or 2'-linear modified nucleoside comprises a sugar moiety comprising a linear 2'-substituent selected from F, OCH ₃ , and OCH ₂ CH ₂ OCH ₃ .

Certain modified sugar moieties include a bridging sugar substituent that forms a second ring, thereby creating a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include, but are not limited to: 4′-CH ₂ -2′, 4′-(CH ₂ ) ₂ -2′, 4′-(CH ₂ ) ₃ -2′, 4′-CH ₂ ―O-2′ ("LNA"), 4′-CH ₂ ―S-2′, 4′-(CH ₂ ) ₂ ―O-2′ ("ENA"), 4′-CH(CH ₃ )―O-2′ (referred to as "constrained ethyl" or "cEt" when in the S configuration), 4′-CH ₂ ―O―CH ₂ -2′, 4′-CH ₂ ―NI-2′, 4′-CH(CH ₂ OCH ₃ )―O-2′ ("constrained MOE" or "cMOE") and analogs thereof (e.g., U.S. Pat. 7,399,845), 4′-C(CH ₃ )(CH ₃ )―O-2′ and analogs thereof (see, e.g., WO2009/006478), 4′-CH ₂ ―N(OCH ₃ )-2′ and analogs thereof (see, e.g., WO2008/150729), 4′-CH ₂ ―O―N(CH ₃ )-2′ (see, e.g., US2004/0171570), 4′-CH ₂ ―C(H)(CH ₃ )-2′ (see, e.g., Chattopadhyaya, et al., J. Org. Chem ., 2009, 74, 118-134), 4′-CH ₂ ―C( CH ₂ )-2′ and analogs thereof (see published PCT International Application WO 2008/154401), 4′-C(R _a R _b I(R)―O-2′, 4′-C(R _a R _I O―N(R)-2′, 4IH ₂ ―O―N(R)-2′, I 4′-CH ₂ ―N(R)―O-2′, wherein each R, R _a , and R _b is independently H, a protecting group, or C ₁ -C ₁₂ alkyl (see, e.g., U.S. Pat. No. 7,427,672).

In certain embodiments, such 4' to 2' bridges independently comprise 1 to 4 linked groups independently selected from: ―[C(R _a )(R _b )] _n ―, ―[C(R _a )(R _b )] _n ―O―, ―C(R _a ) C(R _b )―, ―C(R _a ) N―, ―C( NR _a )―, ―C( O)―, ―C( S)―, ―O―, ―Si(R _a ) ₂ ―, ―S( O) _x ―, and ―N(R _a )―; where X is 0, 1, or 2, and n is 1, 2, 3, or 4.

In some embodiments, the bicyclic sugar moiety and the nucleoside comprising such bicyclic sugar moiety are further defined by the isomeric configuration. For example, an LNA nucleoside (described above) may be in the α-L configuration or the β-D configuration. In other embodiments, the modified sugar moiety is a sugar substitute. In certain such embodiments, an oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain embodiments, such modified sugar moieties also include bridging and/or non-bridging substituents as described above. For example, certain sugar substitutes include substitutions at the 4'-sulfur atom and the 2'-position (see, e.g., US2005/0130923) and/or the 5'-position.

In some embodiments, the modification at the 2' position of the ribose group may be a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

In one embodiment, one or more modifications in the modified gRNa comprises a 2'OMe modification, such as a 2'-O-methyl (M) nucleotide, a 2'-O-methyl 3'-phosphorothioate (MS) nucleotide, a 2'-O-methyl 3'-thioPACE (MSP) nucleotide, or a combination thereof.

In some embodiments, the modified sgRNA comprises one or more nucleosides comprising a modified nucleobase. In certain embodiments, the modified sgRNA comprises one or more nucleosides that do not comprise a nucleobase, referred to as an abasic nucleoside.

In some embodiments, the modified nucleobase is selected from a 5-substituted pyrimidine, a 6-azapyrimidine, an alkyl or alkynyl substituted pyrimidine, an alkyl substituted purine, and an N-2, N-6, and O-6 substituted purine. In certain embodiments, the modified nucleobases are selected from the group consisting of 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl(―C≡C―CH ₃ ) uracil, 5-propynylcytosine, 6-azouracil, 6-azoyitosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyryl guanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-extending bases, and fluorinated bases. Additionally, modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by another heterocycle (e.g., 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone).

In some embodiments, the nucleosides of the modified sgRNAs described herein can be linked to each other using any internucleoside linkage. Two major classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. A representative phosphorus-containing internucleoside linkage is a phosphodiester linkage ("P Phosphates, phosphotriesters, methylphosphonates, phosphoramidates and phosphorothioates ("P") containing (also referred to as unmodified or naturally occurring linkages) S") and phosphorodithioate ("HS―P S"). Representative non-phosphorus-containing internucleoside linking groups include methylenemethylimino (―CH ₂ ―N(CH ₃ )―O―CH ₂ ―), thiodiester (―O―C( O)―S―), thionocarbamate (―O―C( O)(NH)―S―); siloxane (―O―SiH ₂ ―O―); and N,N'-dimethylhydrazine (―CH ₂ ―N(CH ₃ )―N(CH ₃ )―). Modified internucleoside linkages compared to naturally occurring phosphate linkages can be used to modify and typically increase the nuclease resistance of oligonucleotides. In certain embodiments, internucleoside linkages having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free internucleoside linkages are well known to those skilled in the art.

Neutral internucleoside linkages include, without limitation, phosphotriester, methylphosphonate, MMI(3′-CH ₂ ―N(CH ₃ )―O-5′), amide-3(3′-CH ₂ ―C( O)―N(H)-5′), amide-4(3′-CH ₂ ―N(H)―C( Additional neutral internucleoside linkages include, but are not limited to, 3′-O—CH ₂ —O-5′), formacetal (3′-O—CH 2 —O-5′), methoxypropyl, and thioformacetal (3′-S—CH ₂ —O-5′). Additional neutral internucleoside linkages include nonionic linkages involving siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, e.g., Carbohydrate Modifications in Antisense Research ; YS Sanghvi and PD Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, pp. 40-65). Additional neutral internucleoside linkages include nonionic linkages involving mixed N, O, S, and CH ₂ moieties.

In some embodiments, one or more chemical modifications at the phosphate group may be a phosphorothioate modification.

In some embodiments, the modified sgRNA comprises one modified nucleotide at the 5'-end (e.g., at the terminal nucleotide at the 5'-end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5'-end) of the modified gRNA nucleotide sequence and one modified nucleotide at the 3'-end (e.g., at the terminal nucleotide at the 3'-end) or near the 3'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3'-end) of the modified gRNA nucleotide sequence.

In some embodiments, the modified sgRNA comprises two consecutive or non-contiguous modified nucleotides starting at the 5'-end (e.g., at the terminal nucleotide at the 5'-end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5'-end) of the modified gRNA nucleotide sequence and/or two consecutive or non-contiguous modified nucleotides starting at the 3'-end (e.g., at the terminal nucleotide at the 3'-end) or near the 3'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3'-end) of the modified gRNA nucleotide sequence.

Other areas and variations

In some embodiments, the modified gRNA comprises a modified nucleotide at its 5' end. In some embodiments, the 5' end of the sgRNA comprises a spacer or guide region that functions to direct a Cas protein, e.g., a Cas9 protein, to a target nucleotide sequence. In some embodiments, the 5' end does not comprise a guide region. In some embodiments, the 5' end comprises a spacer and additional nucleotides that function to direct the Cas protein to the target nucleotide region.

The sgRNA has a 3' end, which is the last nucleotide of the sgRNA. The 3' end region comprises the last 1 to 7 nucleotides from the 3' end. In some embodiments, the 3' end is the end of hairpin 2. In some embodiments, the sgRNA comprises nucleotides after the hairpin region(s). In some embodiments, the sgRNA comprises a 3' tail region, in which case the last nucleotide of the 3' tail is the 3' terminus. In some embodiments, the 3' tail comprises, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides that are not associated with the secondary structure of the hairpin. In some embodiments, the 3' tail region comprises 1, 2, 3, or 4 nucleotides that are not associated with the secondary structure of the hairpin. In some embodiments, the 3' tail region comprises 4 nucleotides that are not associated with the secondary structure of the hairpin. In some embodiments, the 3' tail region comprises 1, 2, or 3 nucleotides that are not associated with the secondary structure of the hairpin.

In some embodiments, the modified gRNA further comprises a target nucleic acid sequence complementary to the sequence of the target nucleic acid. The complementary sequence is about 20 nucleotides in length. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in the complementary nucleotide sequence are modified nucleotides. In certain cases, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the nucleotides in the complementary nucleotide sequence (e.g., a first nucleotide sequence that is about 20 nucleotides in length) are modified nucleotides. In other cases, all nucleotides within the complementary nucleotide sequence (e.g., a complementary nucleotide sequence that is about 20 nucleotides in length) are modified nucleotides. In some cases, the modified nucleotide is located at the 5'-end (e.g., at the terminal nucleotide at the 5'-end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5'-end) of the complementary nucleotide sequence and/or at an internal position within the complementary nucleotide sequence. In other cases, about 10% to about 30% of the nucleotides of the first nucleotide sequence are modified nucleotides.

Exemplary gRNA sequences

In some embodiments, the present disclosure provides LONGEST gRNAs. Table 1 includes some exemplary LONGEST gRNAs with chemical modifications.

LONGEST design with chemically modified nucleotides designation order LONGEST 1 mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAA GGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU
(Sequence number: 51) LONGEST 2 mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmGmCmGmGmAmAmAmCmGmCmGmCAAGUUAAAAUAAGGCUAGU CCGUUAUCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 52) LONGEST 3 mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmUmGmCmGmAmAmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGC UAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 20) LONGEST 4 mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmUmGmCmUmGmGmAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUA AGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 21)

N = nucleotide; mN = 2'-OMe modified nucleotide; Ns = phosphorothioate nucleotide

Exemplary guide RNA sequences Sequence (5'-3') GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 19) GUUUUAGAGCUAN _x1-n GAAANy _1-n UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU(SEQ ID NO: 42)yn(n=2 to 10)
xn(n=2 to 10) GUUUUAGAmGmCmUmAm _{( N} 43)
yn(n=2 to 10)
xn(n=2 to 10) GUUUUAGAN _xn GAAANy _n AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(Hairpin2) UUUU(SEQ ID NO: 23)
yn(n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
xn(n=, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) GUUUUAGA m ( _N
yn(n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
xn(n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) GUUUUAGAGCGCGGAAACGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 8) GUUUUAGAmGmCmGmCmGmGmAmAmAmCmGmCmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 31) GUUUUAGAmGmCmCmGmGmCmGmGmAmAmCmGmCmCmGmGmCAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 44) GUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 45) GUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAU(AAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO. 46) GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmU mUmGmGmAmCmUmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 47) _{GUUUUAGAN ​} AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 49) AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUCGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 50)

N = nucleotide; mN = 2'-OMe modified nucleotide; Ns = phosphorothioate nucleotide

Synthesis of modified guide RNA

The present disclosure also includes methods for synthesizing a modified gRNA as described herein. In some embodiments, a first RNA sequence, which is a trans-activating RNA (tracrRNA), and a second RNA sequence comprising a clustered regularly spaced short palindromic repeat (CRISPR) RNA (crRNA) sequence complementary to a target sequence are provided for synthesizing a modified sgRNA as described herein.

In some embodiments, the first RNA sequence and the second RNA sequence are ligated together. The ligation strategy described herein differs from previously reported chemical ligation strategies used to synthesize synthetic RNAs, including guide RNAs. An advantage of using this segmented synthesis approach is that shorter sections of RNA can be produced with greater purity after purification compared to the full-length gRNA. In this approach, the 5' acceptor is the smallest RNA fragment (approximately 30 to 50 nts) and can therefore be highly purified prior to ligation. The 3' donor is terminated with a phosphate required for synthesis, so only the full-length fragment will be incorporated into the full-length product (i.e., the cleavage is not a substrate).

In some embodiments, the modified gRNA is synthesized using a self-templating approach. A method for synthesizing the modified gRNA comprises providing a first RNA sequence and a second RNA sequence having complementarity, wherein the complementarity allows for base pairing and the formation of a stem loop between the first RNA and the second RNA; ligating the first RNA and the second RNA using a ligation enzyme within the stem loop to produce the modified gRNA. This allows for the formation of an enzymatic ligation of the two RNAs using a helix or other structure formed between the first RNA and the second RNA. In some embodiments, the length and sequence composition of the structure formed between the first RNA and the second RNA are modified to promote non-covalent bonding and to create an optimal ligation site for an enzyme suitable for RNA ligation.

The complementarity between the nucleotide segments of the first RNA sequence and the second RNA sequence can be partial or complete. Complementarity allows base pairing between complementary nucleotides. In the partially complementary region, the mismatched nucleotides form an overhang or loop structure between the first and second RNA sequences. Various modified gRNA structures, such as a longest upper stem, stem loop, lower stem, hairpin, overhang, blunt end, or overhang, can be formed between the first and second RNA sequences based on hybridization between the two RNA sequences.

A variety of ligases can be used in the methods described herein. For example, one or more of T4 RNA ligase 1, T4 RNA ligase 2, RtcB ligase, thermostable 5′ App DNA/RNA ligase, electro-ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, SplintR ligase, E. coli DNA ligase, 9°N DNA ligase, circle ligase, circle ligase II, DNA ligase I, DNA ligase III, and DNA ligase IV can be used. In some embodiments, T4 RNA ligase 1 is used to ligate the first RNA and the second RNA at the termination loop. In some embodiments, T4 RNA ligase 2 is used to ligate the first RNA sequence and the second RNA sequence within the stem formed between the first RNA sequence and the second RNA sequence.

This approach allows for a variety of ligations, including ligation within the terminal loop of a hairpin formed between a first RNA sequence and a second RNA sequence. Various ligases, such as T4 RNA ligase 1, are suitable for ligation within the terminal loop of a hairpin. Another type of ligation possible with this approach is ligation within a duplex formed between a first RNA and a second RNA. Various ligases, such as T4 RNA ligase 2 and DNA ligase, are suitable for ligation within a duplex formed between two RNAs.

In some embodiments, the modified gRNAs of the present disclosure can be synthesized using a splint template approach. In some embodiments, splints are used to produce synthetic RNAs. Using splints allows one or more RNA molecules to be brought into physical proximity for a reaction using the splint as a template. When two or more RNAs need to be linked, using splints facilitates the production of synthetic RNAs, such as the modified sgRNAs described herein. In some embodiments, the splint can be any suitable polymer capable of bringing one or more RNA molecules into close proximity. For example, in some embodiments, the splint is an RNA molecule or a DNA molecule.

In some embodiments, the splint has complementarity to a portion of a first RNA sequence and a second RNA sequence. The complementarity may be partial or complete. Thus, in some embodiments, a method for producing a modified gRNA comprises providing a first RNA comprising a 5' phosphate group; providing a second RNA comprising a free 3'-hydroxyl group; providing an oligonucleotide having partial complementarity to the first RNA and the second RNA, wherein the complementarity of the oligonucleotide allows base pairing with the first and second RNAs; and providing a ligase that catalyzes ligation between the first and second RNAs, thereby producing the gRNA.

In some embodiments, a non-template approach is used to produce the modified sgRNAs described herein. In some embodiments of the non-template approach, a first RNA having a 5' phosphate group (e.g., a 5' monophosphate group) is provided, and a second RNA comprising a blocked 3' terminus (e.g., a blocked 3' OH group) is provided. The purpose of blocking the 3' OH group of the second RNA is to prevent the second RNA from cyclizing via a non-template mechanism when ligation occurs. For example, using such a non-template approach can include a second RNA (e.g., a dideoxynucleotide) comprising a 3' hydroxyl group at the 3' terminal end of a donor molecule that is chemically blocked or removed, and an enzyme (particularly T4 RNA ligase 1) can catalyze the appropriate ligation between the first RNA and the second RNA. In some embodiments, this ligation strategy is performed at high concentrations.

In some aspects, a non-template approach to generating synthetic RNA comprises providing a first RNA comprising a 5'-monophosphate; providing a second RNA comprising a blocked 3' end; and providing a ligase that catalyzes ligation between the first and second RNAs to produce an sgRNA as described herein.

gene editing system

According to the present disclosure, a gene editing system is provided comprising one or more modified sgRNAs described herein. In some embodiments, the system comprises a CRISPR-Cas9 nuclease or a variant thereof.

A variety of naturally occurring or genetically engineered Cas9 nuclease variants can be incorporated into a pre-configured gene editing system.

In some embodiments, the invention provides a gene editing system comprising one or more modified sgRNAs described herein and a CRISPR-Cas9 nuclease or variant thereof.

In some embodiments, the present invention provides a gene editing system comprising one or more modified sgRNAs described herein and a base editor. The base editor may comprise an adenosine deaminase domain or a cytidine deaminase domain. For example, the one or more modified guide RNAs target the base editor to induce an A≫T to G*C change in a target polynucleotide (e.g., a target gene). In one embodiment, the base editor is a fusion protein comprising one or more domains having base editing activity.

In another embodiment, a protein domain having base editing activity is linked to a guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, the domain having base editing activity can deaminate a base in a nucleic acid molecule. In some embodiments, the base editor can deaminate one or more bases in a DNA molecule. In some embodiments, the base editor can deaminate cytosine (C) or adenosine (A) in DNA. In some embodiments, the base editor can deaminate cytosine (C) and adenosine (A) in DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) or a variant thereof. For example, the ABE includes, but is not limited to, ABE8.8, editor variant ABEV1 (pNMG-B2000(ABE8.20 w/ F149Y "v1") + S82T), or ABEV2 (pNMG-B2001(ABE8.20 w/ Y147D,F149Y,T166I,D167N "v2") + S82T). In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. The base editor can, in some cases, be fused to an inhibitor of base excision repair, e.g., a UGI domain or a dISN domain. In other embodiments, the base editor is an abasic base editor.

A detailed description of base editors can be found in PCT patent application publications WO2018/027078, WO2017/070632, WO 2022/204268, and WO2023/114953, the contents of each of which are incorporated herein by reference for all purposes.

Formulations and compositions

The modified sgRNA and gene editing system of the present disclosure can be formulated into a carrier for delivery and administration. In some embodiments, the pharmaceutical formulation comprises a lipid nanoparticle (LNP). In some embodiments, the pharmaceutical formulation comprises at least one modified gRNA of the present disclosure. In other embodiments, the pharmaceutical formulation comprises at least one modified gRNA and a Cas9 protein, a polynucleotide encoding the Cas9 protein, or an mRNA encoding the Cas9 protein.

Lipid nanoparticles (LNPs) are well known for delivering nucleotide and protein cargo and can be used to deliver gRNA, gene editing systems, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, LNPs deliver nucleic acids, proteins, or nucleic acids in combination with proteins. Accordingly, the present disclosure provides a method for delivering any of the gRNA, gene editing systems, compositions, or pharmaceutical formulations disclosed herein to a subject, wherein the gRNA is associated with an LNP. In some embodiments, the gRNA/LNP is also associated with Cas9 or an mRNA encoding Cas9. In some embodiments, the gRNA/LNP is also associated with a base editor or an mRNA encoding a base editor.

In some embodiments, the present invention comprises a composition comprising any one of the disclosed gRNAs and an LNP. In some embodiments, the composition further comprises a Cas9 protein or a variant thereof, or an mRNA encoding the Cas9 protein or a variant thereof.

In some embodiments, the LNP comprises one or more cationic lipids, one or more helper lipids, one or more PEGylated lipids, and one or more cholesterol-derived lipids.

In some embodiments, a composition comprising a modified gRNA and a gene editing system described herein is provided. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises one or more modified sgRNAs described herein.

Compositions comprising any of the gRNA and/or gene editing systems described herein, carriers, excipients, diluents, etc. are encompassed.

In some embodiments, the modified gRNAs, gene editing systems, compositions and formulations disclosed herein are for use in preparing a medicament for treating a disease or disorder.

How to use

The present disclosure provides for the use of a modified gRNA described herein to alter a target nucleic acid in vitro (e.g., in cells cultured in vitro for ex vivo therapy of gene-edited cells or other uses) or the genome within a cell in a subject, such as a human (e.g., for use in in vivo therapy).

In some embodiments, the invention encompasses a method or use of modulating a target nucleic acid molecule, comprising administering or delivering any one or more of the gRNA gene editing systems, compositions, or pharmaceutical formulations described herein.

In some embodiments, the present invention encompasses methods or uses for modulating a target gene, comprising administering or delivering one or more of the gRNA gene editing systems, compositions, or pharmaceutical formulations described herein. In some embodiments, the modulation is editing of the target gene. In some embodiments, the modulation is a change in the expression of a protein encoded by the target gene.

In some embodiments, the method or use results in gene editing. In some embodiments, the method or use results in a double-strand break within the target gene. In some embodiments, the method or use results in an insertion or deletion of a nucleotide within the target gene. In some embodiments, the insertion or deletion of a nucleotide within the target gene results in a frameshift mutation or a premature stop codon resulting in a non-functional protein. In some embodiments, the insertion or deletion of a nucleotide within the target gene results in a knockdown or elimination of target gene expression. In some embodiments, the method or use further comprises delivering a template to the cell, wherein at least a portion of the template is incorporated into the target DNA at or near the site of the double-strand break induced by the nuclease. In some embodiments, the method or use results in a substitution. In some embodiments, the gene modulation is an increase or decrease in gene expression, a change in the methylation state of DNA, or a modification of a histone subunit. In some embodiments, the method or use results in increased or decreased expression of a protein encoded by the target gene.

The efficacy of the gRNA and/or gene editing system can be tested in vitro and in vivo . In some embodiments, the invention comprises one or more of the gRNA editing systems, compositions, or pharmaceutical formulations described herein, wherein the gRNA results in gene regulation when delivered to a cell together with Cas9 or mRNA encoding Cas9. In some embodiments, the efficacy of the gRNA can be measured in vitro or in vivo .

In some implementations, the efficiency of a gRNA in increasing or decreasing target protein expression is determined by measuring the amount of target protein.

In some embodiments, the editing efficiency by a particular gRNA is determined by the editing present at the target site in the genome after delivery of Cas9 and the gRNA. In some embodiments, the editing efficiency by a particular gRNA is measured by next-generation sequencing. In some embodiments, the percentage of editing in the target region of interest is determined. In some embodiments, the total number of sequence reads with insertions or deletions of nucleotides within the target region of interest relative to the total number of sequence reads is measured after delivery of the modified gRNA and/or a system or composition comprising the modified gRNA.

In some embodiments, the activity of the modified gRNA is measured following in vivo administration of LNPs comprising the modified gRNA.

In some embodiments, the in vivo efficacy of a gRNA or composition provided herein is determined by editing efficacy measured in DNA extracted from a tissue (e.g., liver tissue) following administration of the gRNA.

In some embodiments, a method for modulating a target nucleic acid sequence in a cell comprises contacting the cell with a composition comprising a modified sgRNA described herein.

Methods for therapeutic purposes

In some embodiments, any one or more of the gRNA, gene editing system, composition or pharmaceutical formulation described herein is for use in preparing a medicament for treating or preventing a disease or disorder in a subject.

In some embodiments, the invention includes a method of treating or preventing a disease or disorder in a subject, comprising administering any one or more of the gRNA, gene editing system, composition, or pharmaceutical formulation described herein.

In some embodiments, the gRNA, gene editing system, and composition increase editing efficacy at the target site. In some embodiments, the editing efficacy is increased by about 10% to 100% compared to an unmodified gRNA. In some embodiments, the editing efficacy is increased by about 2-1000 fold compared to an unmodified gRNA. In some embodiments, the sgRNA of the invention increases gene editing efficacy by about 2-1000 fold, about 2-100 fold, about 2-50 fold, about 10-50 fold, about 10-20 fold, about 2-10 fold, about 2-5 fold, about 3-8 fold, or about 2-5 fold. As a non-limiting example, the sgRNAs described herein increase gene modification efficacy by 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 550, 600, 650, 700, 750, 800, 900, or 1000-fold.

Kit

In another aspect of the present disclosure, a kit is provided comprising one or more gRNAs, compositions, or pharmaceutical formulations described herein. In some embodiments, the kit further comprises one or more solvents, solutions, buffers, instructions, or desiccants, each independent of the composition or pharmaceutical formulation.

Example

The following examples illustrate specific embodiments of the present invention and are not limiting.

Example 1 : Editing Efficacy of Modified gRNA

In this example, the ALAS1 (delta-aminolevulinic acid synthase 1) gene region is used to evaluate the impact of different hairpin designs on the editing efficiency of guide RNA.

Measurements for in vivo liver editing (Figure 1A) revealed that when the hairpin was extended without modification, editing efficiency decreased. However, when the hairpin was extended with modification, i.e., with a severe modification encompassing all nucleotides, editing efficiency increased.

Three different gRNAs targeting ALAS1 were synthesized (gRNA1 (terminal modification), gRNA2 (standard highly modified), and gRNA3 (highly modified LONGEST hairpin design)) (Fig. 2A). The editing efficacy of each gRNA in the liver was measured in lipid 1 (Fig. 2B) and lipid 2 (Fig. 2C). gRNA3 with the LONGEST design was found to be 2- to 5-fold more potent at a saturating dose (8% editing at 0.005 mpk) than gRNA2 with the standard highly modified design for ALAS1. The targeting sequences for ALAS1: CAGGATCCGCACAGACTCCAGGG (SEQ ID NO: 59) and the protospacer sequence: CAGGAUCCGCACAGACUCCA (SEQ ID NO: 60) were used in the study.

Examples of gRNA designs used in research designation Sequence terminally deformed mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 53) Highly deformed mNsmNsmNsNNNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 54) Not deformed. LONGEST 1 mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAGCCGGCGGAAACGCCGGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 55)
(2'OMe hairpin extension without modified base) gRNA 4 mAsmUsmAsAUAGCUGGCAUCACGGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 56)
(Standard terminal variant) gRNA 5 mAsmUsmAsAUAGCUGGCAUCACGGUGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 57)
(GOLD modified gRNA) gRNA6 mAsmUsmAsAUAGCUGGCAUCACGGUGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCU AGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 58)
(LONGEST + GOLD modified gRNA)

N = nucleotide; mN = 2'-OMe modified nucleotide; Ns = phosphorothioate nucleotide

The terminally modified gRNA is a standard gRNA design used in the prior art (Hendel, Ayal, et al., Nature biotechnology 33.9 (2015): - 3290). A standard highly modified gRNA without an extended upper stem region is also used for comparison (Finn, Jonathan D., et al., Cell reports 22.9 (2018): 2227-2235). Both the terminally modified and highly modified gRNAs are 100 nucleotides in length. In the LONGEST1 design, the hairpin is extended by an additional 3 base pairs compared to the terminally modified gRNA. The unmodified LONGEST1 has the same sequence as LONGEST 1 (SEQ ID NO: 51) but is not internally modified with 2'OMe. It is 106 nt in length.

Riesenberg et al. (Riesenberg, et al., Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage." Nature Communications 13.1 (2022): 1-8) recently reported that engineered gRNAs with "locked" hairpins can increase the stability and editing efficacy of engineered gRNAs. They compared the LONGEST hairpin with Riesenberg's GOLD (genome editing locking design) hairpin. The in vitro editing efficacy of each gRNA design (common end modification (EM), LONGEST, GOLD, and a combination of LONGEST and GOLD) was measured at three different target sites (TSBTx3288, TSBTx3215, and TSBTx3222) (see Figure 3A). Bold nucleotides in each design represent backbone-modified nucleotides (Figure 3A). Figure 3B shows that both the LONGEST and GOLD designs outperformed gRNAs with end modifications. (EM shown in Figure 3A). An approximately 7-fold increase was observed in the GOLD design compared to the terminal strain design. An approximately 3-fold increase was observed in the LONGEST design compared to the terminal strain design.

We synthesized a gRNA (LONGEST-GOLD) with both LONGEST and GOLD designs and evaluated its editing efficacy (gRNA 6 in Figure 4A). The GOLD-LONGEST and GOLD designs Three different adenine base editors (ABEs) (ABE8.8, ABE9V1, and ABE9V2) were evaluated. ABEV1 used herein is the editing variant pNMG-B2000 (ABE8.20 w/ F149Y "v1") + S82T. ABEV2 is the editing variant pNMG-B2001 (ABE8.20 w/ Y147D, F149Y, T166I, D167N "v2") + S82T.

Editing efficacy after 5 days shows that hairpin extension increases overall editing of the gRNA under saturating conditions (Figure 4B).

Example 2: New extension design

In this study, we additionally tested various hairpin extensions of gRNA. Three novel hairpin extensions with lower GC content than the LONGEST variant promote hairpin stability. The sequences and modifications of the three gRNA designs are shown in Table 4. LONGEST 3 has three additional base pairs with a complete modification of the upper stem loop. LONGEST 4 has five additional base pairs with a complete modification. Unmodified LONGEST 4 has the same extension as LONGEST 4 but without modification.

designation order LONGEST 3 mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmUmGmCmGmAmAmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGC UAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 20) LONGEST 4 mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmUmGmCmUmGmGmAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUA AGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 21) Undeformed LONGEST mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 22)
(Hairpin extension without modified base)

The three novel hairpin designs were tested for in vivo editing efficacy using the base editor ABE8.8, compared to the standard high-altitude and terminal-altitude modification designs and the LONGEST design targeting ALAS1. The results show that LONGEST 4 exhibits higher efficacy than the standard high-altitude and terminal-altitude modification designs (Figure 5). Unmodified LONGEST 4 exhibited lower efficacy, suggesting that extension alone is sufficient and that modification is necessary to enhance efficacy. Similar results were observed with the LONGEST design.

Equivalents and Scope

Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not limited to the above description, but is as set forth in the following claims:

Attach an electronic file of the sequence list

Claims (75)

A single guide RNA (sgRNA) comprising a long (or extended) upper stem comprising four or more base pairs formed by complementary nucleotides, wherein at least one nucleotide of the upper stem is a modified nucleotide. In the first paragraph, the long upper stem region comprises 5 to 15 base pairs formed by complementary nucleotides. In the second paragraph, the sgRNA, wherein the long upper stem region comprises five base pairs formed by complementary nucleotides. In the second paragraph, the sgRNA, wherein the long upper stem region comprises six base pairs formed by complementary nucleotides. In the second paragraph, the sgRNA, wherein the long upper stem region comprises seven base pairs formed by complementary nucleotides. In the second paragraph, the sgRNA, wherein the long upper stem region comprises eight base pairs formed by complementary nucleotides. In the second paragraph, the sgRNA, wherein the long upper stem region comprises 9 base pairs formed by complementary nucleotides. In the second paragraph, the sgRNA, wherein the long upper stem region comprises 10 base pairs formed by complementary nucleotides. An sgRNA according to any one of claims 1 to 8, wherein all nucleotides of the upper stem are modified nucleotides. An sgRNA according to any one of claims 1 to 9, wherein at least one nucleotide within the hairpin 1 and hairpin 2 regions is a modified nucleotide. In claim 10, an sgRNA wherein all nucleotides within the hairpin 1 and 2 regions are modified nucleotides. An sgRNA according to any one of claims 1 to 11, wherein the sgRNA further comprises a modified stable hairpin 1 region. In claim 12, the modified stable hairpin 1 region comprises an extended stem region comprising at least 4 base pairs, and the loop of hairpin 1 comprises a locked nucleic acid. A guide RNA comprising an extended upper stem comprising at least four base pairs formed by complementary nucleotides, wherein the nucleotides in the upper stem comprise a 2'-O methyl modification. A single guide RNA (sgRNA) comprising the sequence of GUUUUAGA N _xn GAAA Ny _n AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 1),
A single guide RNA (sgRNA), wherein N _xn and N _yn are complementary nucleotides having the same number of nucleotides and forming base pairs, the nucleotides of N _xn and N _yn are modified nucleotides, and n is an integer from 5 to 15. In claim 15, wherein Nx and Ny are each complementary nucleotides and comprise 5 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N _x N _x GAAAN _y N _y N _y N _{y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 2). In claim 15, wherein Nx and Ny are each complementary nucleotides and comprise 6 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N x N _x N _{x GAAAN y N y} N y N _y N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 3). In claim 15, wherein Nx and Ny are each complementary nucleotides and comprise 7 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N x N _x N _x N _x GAAAN _{y N y N y N} y N _{y N y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4). In claim 15, wherein Nx and Ny are each complementary nucleotides and comprise 8 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N x N _x N _x N _x N _x GAAAN _{y N y N y} N _y N _y N _y N _{y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 5). In claim 15, wherein Nx and Ny are each complementary nucleotides and comprise 9 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N x N _x N _x N x N _{x N x} N _x N _x GAAAN _{y N y N y} N _y N y N _y N _{y N y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 6). In claim 15, wherein Nx and Ny are each complementary nucleotides and comprise 10 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N x N _x N x N _x N _{x N x N x N x} GAAAN _{y N y N y} N _y N y N _y N _{y N y} N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 7). In claim 15, the sgRNA comprises a sequence of GUUUUAGAGCGCGGAAACGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 8). In claim 14, the sgRNA comprises a sequence of GUUUUAGAGCCGGCGGAAACGCCGGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 9). An sgRNA according to any one of claims 14 to 23, wherein all nucleotides in the upper stem comprise a 2'O-methyl modification. In claim 24, the sgRNA comprises a sequence set forth in any one of SEQ ID NOs: 20 to 32 and 51 to 52. An sgRNA according to any one of claims 14 to 25, wherein at least one nucleotide within the hairpin 1 and hairpin 2 regions is a modified nucleotide. In claim 26, an sgRNA wherein all nucleotides within the hairpin 1 and hairpin 2 regions are modified nucleotides. An sgRNA according to any one of claims 14 to 27, wherein the loop of the hairpin 1 comprises a 2'-O-methyl modified nucleotide (e.g., 2'-O-methyl 3'-phosphorothioate (MS) nucleotide, 2'-O-methyl 3'-thioPACE (MSP) nucleotide), a 2'-F modified nucleotide, a locked nucleic acid, MOE (methoxyethyl), a DNA nucleotide functionalized for conjugation, and a combination thereof. A single guide RNA (sgRNA) comprising the sequence of GUUUUAGAN _xn GAAAN _yn AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 10),
A single guide RNA (sgRNA), wherein N _xn and N _yn are complementary nucleotides having the same number of nucleotides and forming base pairs, the nucleotides of N _xn and N _yn are modified nucleotides, and n is an integer from 5 to 15. In claim 29, wherein Nx and Ny are each complementary nucleotides and comprise 5 nucleotides forming the upper stem region of the sgRNA, and the sgRNA is GUUUUAGAN _x N _x N _x N _x N _x GAAAN _y N _{y N y N y} An sgRNA comprising the sequence of AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 11). In claim 29, wherein Nx and Ny are each complementary nucleotides and comprise 6 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N x N _x N _x GAAAN _{y N y} N _y N _y N _{y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 12). In claim 29, wherein Nx and Ny are each complementary nucleotides and comprise 7 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N x N _x N _x N _x GAAAN _{y N y N y N y N y} N _{y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 13). In claim 29, wherein Nx and Ny are each complementary nucleotides and comprise 8 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N x N _x N _x N _x N _{x N x} GAAAN _{y N y N y} N _y N y N _y N _{y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 14). In claim 29, wherein Nx and Ny are each complementary nucleotides and comprise 9 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N x N _x N _x N x N _{x N x N x N} x GAAAN _{y N y N y} N y N y N _y N _{y N y N y} AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 15). In claim 29, wherein Nx and Ny are each complementary nucleotides and comprise 10 nucleotides forming the upper stem region of the sgRNA, and the sgRNA comprises a sequence of GUUUUAGAN _x N _x N _x N x N _x N x N _x N _{x N x N x N x} GAAAN _{y N y N y N y} N y N _y N _{y N y} N _y N _y AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 16). In claim 29, the sgRNA comprises a sequence of GUUUUAGAGCCGGCGGAAACGCCGGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUCGGUCCAAGUUUUU (SEQ ID NO: 17). A single guide RNA (sgRNA) comprising the sequence of GUUUUAGAN _xn GAAAN _yn AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACN _z N _z N _z N _z GUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 18),
A single guide RNA (sgRNA) wherein N _xn and N _yn are complementary nucleotides having the same number of nucleotides and forming a base pair, the nucleotides of N _xn and N _yn are modified nucleotides, n is an integer from 5 to 15, and four nucleotides of the loop of the hairpin 1 (N _z N _z N _z N _z ) comprise a sequence of UUCG, CUUG, or GCAA. An sgRNA according to any one of claims 29 to 37, wherein at least one nucleotide within the hairpin 1 and hairpin 2 regions is a chemically modified nucleotide. In claim 38, an sgRNA wherein all nucleotides within the hairpin 1 and hairpin 2 regions are modified nucleotides. An sgRNA according to any one of claims 29 to 39, wherein the loop of hairpin 1 comprises a locked nucleic acid. An sgRNA according to any one of claims 1 to 40, wherein the modification comprises a 2'-O-methyl modified nucleotide (e.g., 2'-O-methyl 3'-phosphorothioate (MS) nucleotide, 2'-O-methyl 3'-thioPACE (MSP) nucleotide), a 2'-F modified nucleotide, a locked nucleic acid, MOE (methoxyethyl), a DNA nucleotide functionalized for conjugation, and a combination thereof. An sgRNA according to any one of claims 1 to 41, wherein at least one modification comprises a 2'-O'methyl (2'-O-Me) modified nucleotide. An sgRNA according to any one of claims 29 to 42, wherein all nucleotides in the upper stem comprise a 2'O-methyl modification. In claim 43, the sgRNA comprises a sequence set forth in any one of SEQ ID NOs: 33 to 40. An sgRNA according to any one of claims 1 to 44, wherein the gRNA further comprises a spacer sequence at the 5' end of the sgRNA; wherein the spacer sequence comprises a sequence complementary to a target sequence of interest. In claim 45, the spacer sequence comprises about 18 to 25, or 18 to 30, or 20 to 25, or 20 to 30 nucleotides. An sgRNA according to any one of claims 1 to 46, wherein the 3' end of the sgRNA is modified. An sgRNA according to any one of claims 1 to 47, wherein the 5' end of the sgRNA is modified. In claim 48, an sgRNA, wherein at least the first three nucleotides at the 5' end of the sgRNA are modified nucleotides. An sgRNA according to any one of claims 1 to 46, wherein the sgRNA comprises a modification at the 5' end of the sequence and a modification at the 3' end of the sequence. An sgRNA according to any one of claims 1 to 50, wherein about 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 75%, 80%, 90%, or 100% of the nucleotides of the sgRNA are modified nucleotides. An sgRNA according to any one of claims 1 to 51, wherein the sgRNA further comprises a nuclear localization sequence (NLS). An sgRNA according to any one of claims 1 to 52, wherein the sgRNA comprises about 102 to 150 nucleotides. In claim 53, the sgRNA comprises about 102 to 120 nucleotides. An sgRNA according to any one of claims 1 to 54, wherein the sgRNA increases the gene modification efficacy by about 2 to 1000 times. In claim 55, the sgRNA increases the gene modification efficacy by about 2 to 100 times, 2 to 50 times, 2 to 20 times, or 10 to 50 times. In claim 56, the sgRNA increases the gene modification efficacy by about 2 to 10 times, 2 to 5 times, 3 to 8 times, or 5 to 10 times. As a genetic modification system,
(a) a CRISPR-associated protein (Cas) polypeptide, or a variant thereof; and
(b) A genetic modification system comprising a single guide RNA (sgRNA) according to any one of claims 1 to 57. A genetic modification system in claim 58, wherein the Cas polypeptide is a Cas9 protein. A genetic modification system according to claim 59, wherein the Cas9 is S. pyogenes Cas9 or S. aureus Cas9. A genetic modification system in claim 60, wherein the Cas9 polypeptide is a nickase or dCas9. A genetic modification system according to any one of claims 58 to 61, wherein the genetic modification efficacy is increased by about 2 to 1000 times. A genetic modification system in claim 62, wherein the genetic modification efficacy is increased by about 2 to 100 times, or 2 to 50 times, or 2 to 20 times, or 10 to 50 times. A genetic modification system in claim 63, wherein the genetic modification efficacy is increased by about 2 to 10 times, or 2 to 5 times, or 3 to 8 times, or 5 to 10 times. A composition comprising a single guide RNA of any one of claims 1 to 59. A composition according to claim 65, further comprising a Cas9 polypeptide or a variant thereof. A composition according to claim 66, wherein the Cas polypeptide is a Cas9 protein, dCas9 or nickcase. A composition according to any one of claims 65 to 67, wherein the composition is formulated as a lipid nanoparticle. A method for modifying a target gene in a cell, comprising the step of introducing into the cell a gene editing system comprising a single guide RNA (sgRNA) of any one of claims 1 to 59. In claim 66, a CRISPR-associated protein (Cas) polypeptide or a variant thereof is introduced into a cell,
A method further comprising the step of guiding a Cas polypeptide to the target gene, wherein the sgRNA induces modification of the target gene with improved activity compared to a corresponding unmodified sgRNA comprising 5'GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU 3' (SEQ ID NO: 19). A method according to claim 70, wherein the cell is a Cas9 expressing cell. A method according to any one of claims 70 to 71, wherein the cell is a mammalian cell. A method according to any one of claims 70 to 72, wherein the cell is an immune cell. A kit comprising the sgRNA of any one of claims 1 to 57, the genetic modification system of any one of claims 58 to 64, or the composition of any one of claims 65 to 68. A single guide RNA comprising a sequence set forth in any one of SEQ ID NOs: 20 to 21 and 51 to 52.