Engineered CRISPR-Cas9 nucleases

Patent metadata

Field	Value
Publication number	US-10633642-B2
Application number	US-201816155808-A
Country	US
Kind code	B2
Filing date	Oct 9, 2018
Priority date	Aug 28, 2015
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

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. A Streptococcus pyogenes Cas9 (SpCas9):single guide RNA (sgRNA) complex comprising a SpCas9 protein with mutations at one or both of Q695 and Q926 in SEQ ID NO: 1, and a sgRNA.

2. The SpCas9:sgRNA complex 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 SpCas9:sgRNA complex of claim 1 , wherein the SpCas9 protein comprises all four of the following mutations: N497A, R661A, Q695A, and Q926A.

4. The SpCas9:sgRNA complex of claim 3 , wherein the SpCas9 protein further comprises mutations at one, two, three, four, or all five of L169, Y450, N497, R661, and D1135.

5. The SpCas9:sgRNA complex of claim 1 , wherein the SpCas9 protein comprises 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 SpCas9:sgRNA complex of claim 1 , wherein the SpCas9 protein further comprises 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 SpCas9:sgRNA complex of claim 1 , wherein the SpCas9 protein further comprises 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 SpCas9:sgRNA complex of claim 7 , wherein the mutations that decrease nuclease activity are: (i) D10A or D10N, and (ii) H840A, H840N, or H840Y.

9. The SpCas9:sgRNA complex of claim 1 , wherein the SpCas9 protein is 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 SpCas9:sgRNA complex of claim 9 , wherein the heterologous functional domain is a transcriptional activation domain.

11. The SpCas9:sgRNA complex of claim 10 , wherein the transcriptional activation domain is from VP64 or NF-κB p65.

12. The SpCas9:sgRNA complex of claim 9 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.

13. The SpCas9: sgRNA complex 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 SpCas9:sgRNA complex of claim 12 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

15. The SpCas9:sgRNA complex of claim 9 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

16. The SpCas9:sgRNA complex of claim 15 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein.

17. The SpCas9:sgRNA complex of claim 16 , wherein the TET protein is TET1.

18. The SpCas9:sgRNA complex of claim 9 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

19. The SpCas9:sgRNA complex 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 SpCas9:sgRNA complex of claim 9 , wherein the heterologous functional domain is a biological tether.

21. The SpCas9:sgRNA complex of claim 20 , wherein the biological tether is MS2, Csy4 or lambda N protein.

22. The SpCas9:sgRNA complex of claim 9 , wherein the heterologous functional domain is FokI.

23. A vector encoding the SpCas9 protein and the sgRNA of claim 1 .

24. A host cell comprising the nucleic acid of claim 23 .

25. A method of altering the genome of a cell, the method comprising expressing in the cell or contacting the cell with the SpCas9:sgRNA complex of claim 1 , wherein the sgRNA has a region complementary to a selected portion of the genome of the cell, whereby the genome of the cell is altered.

26. The method of claim 25 , wherein the SpCas9 protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.

27. A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the SpCas9:sgRNA complex of claim 1 , wherein the sgRNA has 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

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N9/22 Primary

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

• C12N15/902

  using homologous recombination · CPC title

• C12N15/907

  in mammalian cells · CPC title