IP Library › Granted Patent US 12,435,331
Granted Patent B2
US 12,435,331 · App. 18/059,308 · Granted Oct 7, 2025

Cytosine to guanine base editor

Inventors: David R. Liu (Cambridge, MA); Luke W. Koblan (Cambridge, MA)
Assignee: President and Fellows of Harvard College
C12N15/102C07K14/47C12N9/22C12N9/2497C12N9/78C07K2319/80C12Y302/02027C12Y305/04005
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Quick Facts
Patent No.
US 12,435,331
App. No.
18/059,308
Filed
Nov 28, 2022
Granted
Oct 7, 2025
Kind
B2
Art Unit
1656
USPC
435/91.1
Abstract

Some aspects of this disclosure provide compositions, strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins capable of inducing a cytosine (C) to guanine (G) change in a nucleic acid (e.g., genomic DNA) are provided. In some embodiments, fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9) and nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9), and nucleic acid editing proteins or domains, are provided.

Claims (77)

1. A polynucleotide encoding a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the napDNAbp domain when in association with a guide RNA (gRNA) specifically binds a target nucleic acid molecule; (ii) a cytidine deaminase domain, wherein the cytidine deaminase domain deaminates a cytosine base in the target nucleic acid molecule; and (iii) a uracil binding protein (UBP), wherein the UBP is a uracil DNA glycosylase (UDG) or a uracil base excision enzyme; wherein the fusion protein comprises the structure:

NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-COOH,

NH 2 -[cytidine deaminase domain]-[UBP]-[napDNAbp domain]-COOH;

NH 2 -[UBP]-[cytidine deaminase domain]-[napDNAbp domain]-COOH;

NH 2 -[UBP]-[napDNAbp domain]-[cytidine deaminase domain]-COOH;

NH 2 -[napDNAbp]-[UBP]-[cytidine deaminase]-COOH; or

NH 2 -[napDNAbp]-[cytidine deaminase]-[UBP]-COOH;

wherein each instance of “]-[” comprises an optional linker.

2. The polynucleotide of claim 1 , wherein the uracil binding protein is a uracil base excision enzyme.

3. The polynucleotide of claim 1 , wherein the uracil binding protein is a uracil DNA glycosylase (UDG).

4. The polynucleotide of claim 1 , wherein the uracil binding protein (UBP) comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 48 (UDG), SEQ ID NO: 49 (UdgX), SEQ ID NO: 50 (UdgX*), SEQ ID NO: 51 (UdgX_On), or SEQ ID NO: 53 (SMUG1).

5. The polynucleotide of claim 1 , wherein the fusion protein comprises the structure:

NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-COOH,

wherein each instance of “]-[” comprises an optional linker.

6. The polynucleotide of claim 1 , wherein the fusion protein further comprises (iv) a nucleic acid polymerase domain (NAP), wherein the fusion protein comprises the structure:

NH 2 -[NAP]-[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-COOH;

NH 2 -[cytidine deaminase domain]-[NAP]-[napDNAbp domain]-[UBP]-COOH;

NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[NAP]-[UBP]-COOH;

NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-[NAP]-COOH;

NH 2 -[NAP]-[cytidine deaminase domain]-[UBP]-[napDNAbp domain]-COOH;

NH 2 -[cytidine deaminase domain]-[NAP]-[UBP]-[napDNAbp domain]-COOH;

NH 2 -[cytidine deaminase domain]-[UBP]-[NAP]-[napDNAbp domain]-COOH;

NH 2 -[cytidine deaminase domain]-[UBP]-[napDNAbp domain]-[NAP]-COOH;

NH 2 -[NAP]-[UBP]-[cytidine deaminase domain]-[napDNAbp domain]-COOH;

NH 2 -[UBP]-[NAP]-[cytidine deaminase domain]-[napDNAbp domain]-COOH;

NH 2 -[UBP]-[cytidine deaminase domain]-[NAP]-[napDNAbp domain]-COOH;

NH 2 -[UBP]-[cytidine deaminase domain]-[napDNAbp domain]-[NAP]-COOH;

NH 2 -[NAP]-[UBP]-[napDNAbp domain]-[cytidine deaminase domain]-COOH;

NH 2 -[UBP]-[NAP]-[napDNAbp domain]-[cytidine deaminase domain]-COOH;

NH 2 -[UBP]-[napDNAbp domain]-[NAP]-[cytidine deaminase domain]-COOH;

NH 2 -[UBP]-[napDNAbp domain]-[cytidine deaminase domain]-[NAP]-COOH;

NH 2 -[NAP]-[napDNAbp]-[UBP]-[cytidine deaminase]-COOH;

NH 2 -[napDNAbp]-[NAP]-[UBP]-[cytidine deaminase]-COOH;

NH 2 -[napDNAbp]-[UBP]-[NAP]-[cytidine deaminase]-COOH;

NH 2 -[napDNAbp]-[UBP]-[cytidine deaminase]-[NAP]-COOH;

NH 2 -[NAP]-[napDNAbp]-[cytidine deaminase]-[UBP]-COOH;

NH 2 -[napDNAbp]-[NAP]-[cytidine deaminase]-[UBP]-COOH;

NH 2 -[napDNAbp]-[cytidine deaminase]-[NAP]-[UBP]-COOH; or

NH 2 -[napDNAbp]-[cytidine deaminase]-[UBP]-[NAP]-COOH;

wherein each instance of “]-[” comprises an optional linker.

7. The polynucleotide of claim 6 , wherein the nucleic acid polymerase domain has translesion polymerase activity.

8. The polynucleotide of claim 6 , wherein the nucleic acid polymerase domain is from Rev7, Rev1 complex, polymerase iota, polymerase kappa, or polymerase eta.

9. The polynucleotide of claim 6 wherein the fusion protein comprises the structure:

NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-[NAP]-COOH;

NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[NAP]-[UBP]-COOH;

NH 2 -[cytidine deaminase domain]-[NAP]-[napDNAbp domain]-[UBP]-COOH; or

NH 2 -[NAP]-[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-COOH;

wherein each instance of “]-[” comprises an optional linker.

10. The polynucleotide of claim 1 , wherein the napDNAbp domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 4-26.

11. The polynucleotide of claim 1 , wherein the napDNAbp domain is a Cas9 nickase (nCas9).

12. The polynucleotide of claim 1 , wherein the cytidine deaminase domain is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family.

13. The polynucleotide of claim 1 , wherein the cytidine deaminase domain comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of SEQ ID NOs: 67-101.

14. The polynucleotide of claim 1 , wherein the cytidine deaminase domain is a rat APOBEC1 (rAPOBEC1) deaminase comprising one or more mutations selected from the group consisting of W90Y, R126E, and R132E of SEQ ID NO: 93.

15. A polynucleotide encoding a fusion protein comprising:

(i) a first domain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 4-40;

(ii) a second domain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 67-101; and

(iii) a third domain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 48-53.

16. The polynucleotide of claim 1 , wherein the uracil binding protein (UBP) comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 49 (UdgX), SEQ ID NO: 50 (UdgX*), SEQ ID NO: 51 (UdgX_On), or SEQ ID NO: 53 (SMUG1).

17. The polynucleotide of claim 1 , wherein at least one of (i) the cytidine deaminase domain and the napDNAbp domain, and (ii) the napDNAbp domain and the UBP are fused via a linker, and wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 102-109, 120, and 123.

18. A vector comprising the polynucleotide of claim 1 .

19. A cell comprising the polynucleotide of claim 1 .

20. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the polynucleotide of claim 1 ex vivo to a cell from the subject.

21. A kit comprising a nucleic acid construct comprising the polynucleotide of claim 1 further comprising a heterologous promoter that drives expression of the fusion protein.

22. A method of editing a nucleobase pair of a double-stranded DNA sequence in a cell, the method comprising:

contacting the cell with a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, and the polynucleotide of claim 1 in vitro or ex vivo under conditions suitable for expression of the encoded fusion protein in the cell and the formation of a complex in the cell comprising the fusion protein and the guide nucleic acid; thereby:

inducing strand separation of a target nucleic acid molecule; and

excising a cytosine or a thymine in a single strand of the target nucleic acid molecule.

23. The polynucleotide of claim 1 , wherein the uracil binding protein (UBP) comprises the amino acid sequence of any one of SEQ ID NO: 48 (UDG), SEQ ID NO: 49 (UdgX), SEQ ID NO: 50 (UdgX*), SEQ ID NO: 51 (UdgX_On), or SEQ ID NO: 53 (SMUG1).

24. The polynucleotide of claim 6 , wherein the nucleic acid polymerase domain comprises the amino acid sequence of any one of SEQ ID NOs: 54-64.

25. The polynucleotide of claim 1 , wherein the napDNAbp domain comprises the amino acid sequence of any one of SEQ ID NOs: 4-26.

26. The polynucleotide of claim 1 , wherein the cytidine deaminase domain comprises the amino acid sequence of any one of SEQ ID NOs: 67-101.

27. The polynucleotide of claim 15 , wherein:

(i) the first domain comprises the amino acid sequence of any one of SEQ ID NOs: 4-40;

(ii) the second domain comprises the amino acid sequence of any one of SEQ ID NOs: 67-101; and

(iii) the third domain comprises the amino acid sequence of any one of SEQ ID NOs: 48-53.

28. The polynucleotide of claim 1 , wherein the uracil binding protein (UBP) comprises the amino acid sequence of SEQ ID NO: 49 (UdgX).

29. The polynucleotide of claim 1 , wherein the napDNAbp domain is a nuclease inactive Cas9 (dCas9).

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 10, 2023
From: KOBLAN, LUKE W.
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 062649/0379 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 10, 2023
From: LIU, DAVID R.
To: HOWARD HUGHES MEDICAL INSTITUTE
Reel/Frame 062649/0436 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 10, 2023
From: HOWARD HUGHES MEDICAL INSTITUTE
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 062649/0470 →
Continuity (3)
Division 16492553
Provisional Application 62470175 · Mar 10, 2017
Related Publication 20240035017A1 · Feb 1, 2024
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