High specificity genome editing using chemically modified guide RNAs

Patent metadata

Field	Value
Publication number	US-10767175-B2
Application number	US-201715493129-A
Country	US
Kind code	B2
Filing date	Apr 20, 2017
Priority date	Jun 8, 2016
Publication date	Sep 8, 2020
Grant date	Sep 8, 2020

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.

First claim

We 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).

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

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

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).

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

11. 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.

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

13. 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.

14. 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.

15. 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.

16. 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).

17. 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.

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

19. 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.

20. 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.

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

22. 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).

23. 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).

Assignees

• Agilent Technologies Inc

Inventors

• Dellinger Douglas J
• Ryan Daniel E
• Roy Subhadeep
• Sampson Jeffrey R

Classifications

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12N9/222

  Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes · CPC title

• C12N2310/312

  Phosphonates · CPC title

• C12N2310/3125

  Methylphosphonates · CPC title

• C12N2320/10

  in screening processes · CPC title