IP Library › Patent Application 17726290
Patent Application
App. No. 17/726,290

COMPOSITIONS AND METHODS FOR GENE EDITING

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Patent No.
US None
App. No.
17/726,290
Abstract

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

Claims (26)

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.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 13, 2022
From: WEISSMAN, JONATHAN; GILBERT, LUKE A.; NUNEZ, JAMES; CHEN, JIN
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 061415/0738 →