Granted Patent
B2
US 12,241,096 · App. 18/174,294 · Granted Mar 4, 2025
Engineered CRISPR-Cas9 nucleases with altered PAM specificity
Assignee:
The General Hospital Corporation
C12N9/22C12N9/0071C12N9/1007C12N9/1029C12N9/78C12N9/80C12N15/11C12N15/907A61K38/00C07K2319/71C07K2319/80C12N2310/20C12N2800/80
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Abstract
Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.
Claims (33)
1. A base editor protein comprising a spCas9 protein, wherein the spCas9 protein comprises a PAM interacting domain of SEQ ID NO: 1 that has mutations at positions corresponding to D1135, S1136, and G1218 of SEQ ID NO: 1, wherein the mutation at D1135 is M, the mutation at S1136 is Q, and the mutation at G1218 is K.
2. The base editor protein of claim 1 , further comprising a mutation at position corresponding to E1219 of SEQ ID NO:1.
3. The base editor protein of claim 2 , wherein the mutation at position corresponding to E1219 is S or F.
4. The base editor protein of claim 1 , further comprising:
(i) a mutation at a position corresponding to positions selected from the group consisting of D10, E762, D839, H983, and D986 of SEQ ID NO:1; and/or
(ii) a mutation at position corresponding to H840 or N863 of SEQ ID NO:1.
5. The base editor of claim 4 , wherein the mutations are:
(i) D10A or D10N, and/or
(ii) H840A, H840N, or H840Y.
6. The base editor protein of claim 1 , further comprising a mutation at position corresponding to R1335 of SEQ ID NO:1.
7. The base editor protein of claim 1 , further comprising a mutation at position corresponding to T1337 of SEQ ID NO:1.
8. The base editor protein of claim 1 , further comprising a mutation at position corresponding to E1219 of SEQ ID NO:1, a mutation at position corresponding to R1335 of SEQ ID NO:1, and a mutation at position corresponding to T1337 of SEQ ID NO:1.
9. The base editor protein of claim 8 , wherein the mutation at position corresponding to E1219 is S or F, the mutation at position corresponding to R1335 is E, and the mutation at position corresponding to T1337 is R.
10. The base editor protein of claim 9 , further comprising:
(i) a mutation at a position corresponding to positions selected from the group consisting of D10, E762, D839, H983, and D986 of SEQ ID NO: 1; and/or
(ii) a mutation at position corresponding to H840 or N863 of SEQ ID NO:1.
11. The base editor of claim 10 , wherein the mutations are:
(i) D10A or D10N, and/or
(ii) H840A, H840N, or H840Y.
12. The base editor of claim 11 , wherein the mutation is D10A or D10N.
13. A method of altering the genome of a cell, the method comprising expressing in the cell, or contacting the cell with, the base editor of claim 12 , and a guide RNA having a region complementary to a selected portion of the genome of the cell.
14. A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the base editor of claim 12 , and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.
15. A method of editing a nucleotide base in a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the base editor protein of claim 12 , and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.
16. The base editor protein of claim 1 , wherein the protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.
17. The base editor protein of claim 1 , wherein the base editor comprises (i) a cytidine deaminase domain or (ii) an adenosine deaminase domain.
18. The base editor protein of claim 17 , wherein the cytidine deaminase domain is selected from the group consisting of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) deaminase; activation-induced cytidine deaminase (AID); and cytosine deaminase acting on tRNA (CDAT).
19. The base editor protein of claim 17 , wherein the adenosine deaminase domain is selected from the group consisting of adenosine deaminase 1 (ADA1), ADA2; adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).
20. The base editor protein of claim 1 , wherein the SpCas9 has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
21. The base editor protein of claim 20 , wherein the SpCas9 has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1.
22. The base editor protein of claim 21 , wherein the SpCas9 has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1.
23. A method of altering the genome of a cell, the method comprising expressing in the cell, or contacting the cell with, the base editor of claim 1 , and a guide RNA having a region complementary to a selected portion of the genome of the cell.
24. A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the base editor of claim 1 , and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.
25. A method of editing a nucleotide base in a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the base editor protein of claim 1 , and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jul 27, 2023
From: JOUNG, J. KEITH; KLEINSTIVER, BENJAMIN
To: THE GENERAL HOSPITAL CORPORATION
Reel/Frame 064401/0290 →
Continuity (5)
Provisional Application
62641687
· Mar 12, 2018
Provisional Application
62549303
· Aug 23, 2017
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