# US 10,767,175 B2 · High specificity genome editing using chemically modified guide RNAs

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,767,175 B2
- Document ID: US10767175B2
- Status: Granted
- Kind: B2
- Application number: 15/493,129
- Filed: 2017-04-20
- Published: 2017-12-14
- Granted: 2020-09-08
- Earliest claimed: 2016-06-08
- Assignees: Agilent Technologies, Inc.
- Applicants: Agilent Technologies, Inc.
- Inventors: Douglas J Dellinger; Daniel E Ryan; Subhadeep Roy; Jeffrey R Sampson
- Web page: https://patentgrep.com/patent/US10767175B2

## CPC

- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/312: Phosphonates
- C12N 2310/3125: Methylphosphonates
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2320/10: in screening processes
- C12N 2320/50: Methods for regulating/modulating their activity
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to guide RNAs having chemical modifications and their use in CRISPR-Cas systems. The chemically modified guide RNAs have enhanced specificity for target polynucleotide sequences. The present invention also relates to methods of using chemically modified guide RNAs for cleaving or nicking polynucleotides, and for high specificity genome editing.

## Claims

### Claim 1

A synthetic guide RNA comprising:
(a) a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, wherein the target polynucleotide comprises a target sequence adjacent to a PAM site, and (ii) a stem sequence; and
(b) a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence,
wherein the guide sequence consists of 20-N nucleotides, where N is an integer between −10 and 3; and wherein the guide sequence comprises at least one modification located at position 4-N, 5-N, 7-N, 9-N, 10-N or 11-N relative to the 5′-end of the guide sequence, or a combination thereof; wherein the synthetic guide RNA has enhanced specificity for the target sequence compared to a guide RNA without said modification, and wherein said at least one modification is 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-(2-methoxyethyl) (MOE), a 2′-O-(2-methoxyethyl)-3′-phosphorothioate (MOES), a 2′-O-(2-methoxyethyl)-3′-phosphonoacetate (MOEP), or a 2′-O-(2-methoxyethyl)-3′-thiophosphonoacetate (MOESP).

### Claim 2 (depends on 1)

The synthetic guide RNA of claim 1, wherein said guide RNA is a single guide RNA.

### Claim 3 (depends on 1)

The synthetic guide RNA of claim 1, wherein said at least one modification is located at position 5-N or 11-N relative to the 5′-end of the guide sequence, or a combination thereof.

### Claim 4 (depends on 1)

The synthetic guide RNA of claim 1, further comprising at least one modification at the 5′-end, 3′-end, or both ends of said guide RNA.

### Claim 5 (depends on 4)

The synthetic guide RNA of claim 4, wherein said at least one modification at the 5′-end, the 3′-end, or both ends is independently a 2′-O-methyl (M), a phosphorothioate internucleotide linkage (S), a phosphonoacetate internucleotide linkage (P), a thiophosphonoacetate internucleotide linkage (SP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.

### Claim 6 (depends on 1)

The synthetic guide RNA of claim 1, wherein said target polynucleotide is located within the HBB gene, the IL2RG gene, the CLTA gene, or the VEGFA gene.

### Claim 7 (depends on 6)

The synthetic guide RNA of claim 6, wherein the target polynucleotide comprises GCCCCACAGGGCAGTAA (SEQ ID NO:200) of the HBB gene, TAATGATGGCTTCAACA SEQ ID NO:201) of the IL2RG gene, or GAGTGAGTGTGTGCGTG (SEQ ID NO:202) of the VEGFA gene.

### Claim 8 (depends on 6)

The synthetic guide RNA of claim 6, wherein said guide RNA is a single guide RNA and further comprises at least one modification at the 5′-end, 3′-end or both ends of said guide RNA is independently a 2′-O-methyl (M), a phosphorothioate internucleotide linkage (S), a phosphonoacetate internucleotide linkage (P), a thiophosphonoacetate internucleotide linkage (SP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.

### Claim 9

A synthetic crRNA comprising a guide sequence capable of hybridizing to a target polynucleotide that comprises a target sequence adjacent to a PAM site, wherein said guide sequence consists of 20-N nucleotides, wherein N is an integer between −10 and 3; and wherein the guide sequence comprises at least one modification located at position 4-N, 5-N, 7-N, 9-N, 10-N or 11-N relative to the 5′-end of the guide sequence, or a combination thereof; wherein the synthetic crRNA has enhanced specificity for the target sequence compared to a crRNA without said modification, and wherein said at least one modification is a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-(2-methoxyethyl) (MOE), a 2′-O-(2-methoxyethyl)-3′-phosphorothioate (MOES), a 2′-O-(2-methoxyethyl)-3′-phosphonoacetate (MOEP), or a 2′-O-(2-methoxyethyl)-3′-thiophosphonoacetate (MOESP).

### Claim 10 (depends on 9)

The synthetic crRNA of claim 9, further comprising at least one modification at the 5′-end, 3′-end, or both ends of said crRNA.

### Claim 11 (depends on 1)

A method for enhancing the specificity of a CRISPR function, comprising:
selecting a target polynucleotide;
providing at least one synthetic guide RNA of claim 1;
forming a gRNA:Cas protein complex comprising a Cas protein and the synthetic guide RNA; and
contacting the target polynucleotide with the gRNA:Cas protein complex;
wherein said Cas protein is provided as a protein or as a polynucleotide encoding said Cas protein.

### Claim 12 (depends on 11)

The method of claim 11, wherein said guide RNA is a single guide RNA.

### Claim 13 (depends on 11)

The method of claim 11, wherein said guide RNA further comprises at least one modification at the 5′-end, the 3′-end, or both ends of said guide RNA.

### Claim 14 (depends on 13)

The method of claim 13, wherein said at least one modification at the 5′-end, the 3′-end, or both ends is independently a 2′-O-methyl (M), a phosphorothioate internucleotide linkage (S), a phosphonoacetate internucleotide linkage (P), a thiophosphonoacetate internucleotide linkage (SP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.

### Claim 15 (depends on 11)

The method of claim 11, wherein said guide RNA comprises at least one modification selected from a phosphonoacetate internucleotide linkage or a thiophosphonoacetate internucleotide linkage.

### Claim 16 (depends on 11)

The method of claim 11, wherein said guide RNA comprises at least one modification selected from the group consisting of a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-O-methyl-3′-phosphorothioate (MS) and a 2′-O-(2-methoxyethyl) (MOE).

### Claim 17 (depends on 11)

The method of claim 11, wherein said contacting of said polynucleotide target with said gRNA:Cas protein complex is performed in a cell and wherein said forming said complex is performed outside or inside the cell.

### Claim 18 (depends on 17)

The method of claim 17, wherein said Cas protein is a Cas9 protein.

### Claim 19 (depends on 18)

The method of claim 18, wherein said target polynucleotide is located within the HBB gene, the IL2RG gene, the CLTA gene, or the VEGFA gene.

### Claim 20 (depends on 19)

The method of claim 19, wherein the target polynucleotide comprises GCCCCACAGGGCAGTAA (SEQ ID NO:200) of the HBB gene, TAATGATGGCTTCAACA (SEQ NO:201) of the IL2RG gene, or GAGTGAGTGTGTGCGTG (SEQ ID NO:202) of the VEGEA gene.

### Claim 21 (depends on 11)

The method of claim 11, wherein said forming is performed outside of a cell.

### Claim 22 (depends on 1)

The synthetic guide RNA of claim 1, Wherein said at least one modification is a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-(2-methoxyethyl) (MOE), or a 2′-O-(2-methoxyethyl)-3′-phosphorothioate (MOES).

### Claim 23 (depends on 11)

The method of claim 11, wherein said guide RNA comprises at least one modification selected from the group consisting of 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-(2-methoxyethyl) (MOE), and a 2′-O-(2-methoxyethyl)-3′-phosphorothioate (MOES).