Compositions and methods for gene editing

Patent metadata

Field	Value
Publication number	US-2023103909-A1
Application number	US-202217726290-A
Country	US
Kind code	A1
Filing date	Apr 21, 2022
Priority date	Apr 19, 2018
Publication date	Apr 6, 2023
Grant date	—

Abstract

Provided herein are, inter alia, compositions and methods for manipulation of genomes of living organisms.

First claim

1 .- 20 . (canceled).

21 . A method of silencing a target nucleic acid sequence in a cell, wherein the method comprises:
(i) delivering a first polynucleotide encoding a fusion protein to a cell containing the target nucleic acid, the fusion protein comprising: a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel associated box domain; wherein
(1) the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 23; ddCas12a which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 34; dLbCfp1 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 35; or dFnCfp1 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 36;
(2) the Krüppel associated box domain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16; and
(3) the DNA methyltransferase domain comprises a Dnmt3A protein, a Dnmt3L protein, or a Dnmt3A protein and a Dnmt3L protein; wherein the Dnmt3A protein comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 26, and the Dnmt3L protein comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 28; and
(ii) delivering to the cell a second polynucleotide comprising: (a) a DNA-targeting sequence that is complementary to the target nucleic acid sequence; and (b) a binding sequence for the nuclease-deficient RNA-guide DNA endonuclease enzyme.

22 . The method of claim 21 , wherein the Krüppel associated box domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16.

23 . The method of claim 22 , wherein the Krüppel associated box domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 16.

24 . The method of claim 23 , wherein the Krüppel associated box domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 16.

25 . The method of claim 21 , wherein the Dnmt3A protein comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 26.

26 . The method of claim 25 , wherein the Dnmt3A protein comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 26.

27 . The method of claim 26 , wherein the Dnmt3A protein comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 26.

28 . The method of claim 21 , wherein the Dnmt3L protein comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 28.

29 . The method of claim 28 , wherein the Dnmt3L protein comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 28.

30 . The method of claim 29 , wherein the Dnmt3L protein comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 28.

31 . The method of claim 21 , wherein the dCas9 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23.

32 . The method of claim 31 , wherein the dCas9 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 23.

33 . The method of claim 32 , wherein the dCas9 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 23.

34 . The method of claim 21 , wherein the Krüppel associated box domain comprises an amino acid sequence of SEQ ID NO: 16, the Dnmt3A protein comprises an amino acid sequence of SEQ ID NO: 26, the Dnmt3L protein comprises an amino acid sequence of SEQ ID NO: 28 and the dCas9 comprises an amino acid sequence of SEQ ID NO: 23.

35 . The method of claim 34 , wherein the fusion protein comprises, from N-terminus to C-terminus, a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel associated box domain.

36 . The method of claim 34 , wherein the fusion protein comprises, from N-terminus to C-terminus, a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel associated box domain.

37 . The method of claim 21 , wherein the fusion protein further comprises a nuclear localization sequence.

38 . The method of claim 37 , wherein the nuclear localization sequence comprises an amino acid sequence of SEQ ID NO: 25.

39 . The method of claim 21 , wherein the cell is a eukaryotic cell.

40 . The method of claim 21 , wherein the cell is a mammalian cell.

Assignees

• Univ California

Inventors

• CHEN Jin
• Gilbert Luke
• Nunez James
• Weissman Jonathan

Classifications

• C12Y201/01072

  Site-specific DNA-methyltransferase (adenine-specific) (2.1.1.72) · CPC title

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C07K2319/09

  containing a nuclear localisation signal · CPC title

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title