Engineered CRISPR-Cas9 nucleases

Patent metadata

Field	Value
Publication number	US-10093910-B2
Application number	US-201615369533-A
Country	US
Kind code	B2
Filing date	Dec 5, 2016
Priority date	Aug 28, 2015
Publication date	Oct 9, 2018
Grant date	Oct 9, 2018

Abstract

Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genomic engineering, epigenomic engineering, genome targeting, and genome editing.

First claim

What is claimed is:

1. An isolated Streptococcus pyogenes Cas9 (SpCas9) protein that has at least 95% sequence identity to SEQ ID NO: 1, with mutations at one or both of Q695 and Q926, and wherein the protein retains the ability to interact with a guide RNA and target DNA.

2. The isolated protein of claim 1 , wherein the SpCas9 protein is fused to one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.

3. The isolated protein of claim 1 , comprising all four of the following mutations: N497A, R661A, Q695A, and Q926A.

4. The isolated protein of claim 3 , further comprising mutations at one, two, three, four, or all five of L169, Y450, N497, R661, and D1135.

5. The isolated protein of claim 1 , comprising mutations at one or both of Q695 and Q926, and optionally one, two, three, four, or all five of L169, Y450, N497, R661, and D1135.

6. The isolated protein of claim 1 , further comprising one or more of the following mutations: D1135E; D1135V; D1135V/R1335Q/T1337R (VQR variant); D1135E/R1335Q/T1337R (EQR variant); D1135V/G1218R/R1335Q/T1337R (VRQR variant); or D1135V/G1218R/R1335E/T1337R (VRER variant).

7. The isolated protein of claim 1 , further comprising one or more mutations that decrease nuclease activity selected from the group consisting of mutations at D10, E762, D839, H983, or D986; and at H840 or N863.

8. The isolated protein of claim 7 , wherein the mutations that decrease nuclease activity are:
(i) D10A or D10N, and
(ii) H840A, H840N, or H840Y.

9. A fusion protein comprising the isolated protein of claim 1 , fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.

10. The fusion protein of claim 9 , wherein the heterologous functional domain is a transcriptional activation domain.

11. The fusion protein of claim 10 , wherein the transcriptional activation domain is from VP64 or NF-κB p65.

12. The fusion protein of claim 9 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.

13. The fusion protein of claim 12 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

14. The fusion protein of claim 12 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

15. The fusion protein of claim 9 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

16. The fusion protein of claim 15 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein.

17. The fusion protein of claim 16 , wherein the TET protein is TET1.

18. The fusion protein of claim 9 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

19. The fusion protein of claim 18 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase.

20. The fusion protein of claim 9 , wherein the heterologous functional domain is a biological tether.

21. The fusion protein of claim 20 , wherein the biological tether is MS2, Csy4 or lambda N protein.

22. The fusion protein of claim 9 , wherein the heterologous functional domain is FokI.

23. An isolated nucleic acid encoding the protein of claim 1 .

24. A vector comprising the isolated nucleic acid of claim 23 .

25. A host cell comprising the nucleic acid of claim 24 .

26. A method of altering the genome of a cell, the method comprising expressing in the cell or contacting the cell with the isolated protein of claim 1 , linked to a guide RNA having a region complementary to a selected portion of the genome of the cell, whereby the genome of the cell is altered.

27. The method of claim 26 , wherein the isolated protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.

28. A method of altering a double stranded DNA D (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the isolated protein of claim 1 , linked to a guide RNA having a region complementary to a selected portion of the dsDNA molecule, whereby the dsDNA molecule is altered.

29. A method of altering the genome of a cell, the method comprising contacting the dsDNA molecule with the fusion protein of claim 9 , linked to a guide RNA having a region complementary to a selected portion of the dsDNA molecule, whereby the genome of the cell is altered.

30. A method of altering a double stranded DNA D (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the fusion protein of claim 9 , linked to a guide RNA having a region complementary to a selected portion of the dsDNA molecule, whereby the dsDNA molecule is altered.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Kleinstiver Benjamin
• Pattanayak Vikram

Classifications

• C12N15/907

  in mammalian cells · CPC title

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title