Increasing specificity for RNA-guided genome editing

1. A hybrid guide nucleic acid consisting of the sequence:

(SEQ ID NO: 4)

(X 17-20 )GUUUUAGAGCUAUGCUGUUUUG(X N );

(SEQ ID NO: 5)

(X 17-20 )GUUUUAGAGCUA;

(SEQ ID NO: 6)

(X 17-20 )GUUUUAGAGCUAUGCUGUUUUG;

(SEQ ID NO: 7)

(X 17-20 )GUUUUAGAGCUAUGCU;

(SEQ ID NO: 8)

(X 17-20 )GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG

UCCG(X N );

(SEQ ID NO: 9)

(X 17-20 )GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAU

AAGGCUAGUCCGUUAUC(X N );

(SEQ ID NO: 10)

(X 17-20 )GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUA

GCAAGUUAAAAUAAGGCUAGUCCGUUAUC(X N );

(SEQ ID NO: 11)

(X 17-20 )GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG

UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X N );

(SEQ ID NO: 12)

(X 17-20 )GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAG

UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

(SEQ ID NO: 13)

(X 17-20 )GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAA

AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG

GUGC;

and

(SEQ ID NO: 14)

(X 17-20 )GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAA

AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG

GUGC,

wherein the X 17-20 is a sequence complementary to 17-20 consecutive nucleotides of the complementary strand of a target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), wherein one or more of the nucleotides is a deoxyribonucleic acid, and N is 0-50.

2. A vector comprising the DNA molecule of claim 1 .

3. A host cell expressing the hybrid guide nucleic acid of claim 1 .

4. The hybrid guide nucleic acid of claim 1 , wherein the one or more deoxyribonucleotides are within the sequence complementary to 17-20 consecutive nucleotides of the complementary strand of the target sequence.

5. The hybrid guide nucleic acid of claim 1 , wherein the one or more deoxyribonucleotides comprise thymine in place of uracil.

6. The hybrid guide nucleic acid of claim 1 , wherein the X 17 -20 is at least partially or wholly DNA.

7. A composition comprising:

a nucleic acid encoding a variant S. pyogenes Cas 9 protein comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 with mutations at D10, E762, H983, D986, H840 or N863; and

a nucleic acid encoding a hybrid guide nucleic acid that directs the variant S. pyogenes Cas9 protein to a target sequence;

wherein the hybrid guide nucleic acid is selected from the group consisting of:

(X 17 -20) GUUUUAGAGCUAUGCUGUUUUG(XN) (SEQ ID NO:4);

(X 17 -20) GUUUUAGAGCUA (SEQ ID NO:5);

(X 17 -20) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:6);

(X 17 -20) GUUUUAGAGCUAUGCU (SEQ ID NO:7);

(X 17 -20) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(XN) (SEQ ID NO:8);

(X 17 -20) GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUC(XN) (SEQ ID NO:9);

(X 17 -20) GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGUU AAAAUAAGGCUAGUCCGUUAUC(XN) (SEQ ID NO:10);

(X 17 -2o)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC(XN) (SEQ ID NO:11);

(X 17 -2o)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO:12);

(X 17 -2o)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:13); and

(X 17 -2o)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:14),

wherein the X 17 -20 is a sequence complementary to 17-20 consecutive nucleotides of the complementary strand of a target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), wherein one or more of the nucleotides is a deoxyribonucleic acid, and N is 0-50.

8. The composition of claim 7 , wherein the one or more deoxyribonucleotides are within the sequence complementary to 17-20 consecutive nucleotides of the complementary strand of the target sequence.

9. The composition of claim 7 , wherein the one or more deoxyribonucleotides comprise thymine in place of uracil.

10. The composition of claim 7 , wherein the X 17 -20 is at least partially or wholly DNA.

11. The composition of claim 7 , wherein the variant S. pyogenes Cas 9 comprises a mutation at positions D10 and H840.

12. The composition of claim 11 , wherein the mutation at position D10 is D10A or D10N, and the mutation at position H840 is H840A, H840N or H840Y.

13. The composition of claim 7 , wherein the variant S. pyogenes Cas 9 protein is fused to a heterologous functional domain, with an optional intervening linker.

14. The composition of claim 13 , wherein the heterologous functional domain is FokI.

15. The composition of claim 13 , wherein the heterologous functional domain is a transcriptional activation domain.

16. The composition of claim 15 , wherein the transcriptional activation domain is from VP64 or NK- K B p65.

17. The composition of claim 13 , wherein the heterologous functional domain is a transcriptional silencer or a transcriptional repression domain.

18. The composition of claim 17 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

19. The composition of claim 18 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

20. The composition of claim 13 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

21. The composition of claim 20 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) protein.

22. The composition of claim 21 , wherein the TET protein is TET1.

23. The composition of claim 13 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

24. The composition of claim 23 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone methyltransferase (HMT), or a histone demethylase.

25. The composition of claim 13 , wherein the heterologous functional domain is a biological tether.

26. The composition of claim 25 , wherein the biological tether is MS2, CRISPR/Cas Subtype Ypest protein 4 (Csy4), or lambda N protein.

27. The hybrid guide nucleic acid of claim 1 , wherein N is 0-20.

28. The hybrid guide nucleic acid of claim 1 , wherein N is 0.

29. The composition of claim 7 , wherein N is 0-20.

30. The composition of claim 7 , wherein N is 0.