Crispr-based genome modification and regulation

Patent metadata

Field	Value
Publication number	US-2016017366-A1
Application number	US-201314649777-A
Country	US
Kind code	A1
Filing date	Dec 5, 2013
Priority date	Dec 6, 2012
Publication date	Jan 21, 2016
Grant date	—

Abstract

The present invention provides RNA-guided endonucleases, which are engineered for expression in eukaryotic cells or embryos, and methods of using the RNA-guided endonuclease for targeted genome modification in eukaryotic cells or embryos. Also provided are fusion proteins, wherein each fusion protein comprises a CRISPR/Cas-like protein or fragment thereof and an effector domain. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Also provided are methods for using the fusion proteins to modify a chromosomal sequence or regulate expression of a chromosomal sequence.

First claim

1 - 12 . (canceled)

13 . A method for modifying a chromosomal sequence in a eukaryotic cell or embryo, the method comprising:
a) introducing into the eukaryotic cell or embryo (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal, (ii) at least one guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor polynucleotide; and b) culturing the eukaryotic cell or embryo such that each guide RNA directs an RNA-guided endonuclease to a targeted site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break in the targeted site, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified.

14 . The method of claim 13 , wherein the RNA-guided endonuclease is derived from a Cas9 protein.

15 . The method of claim 13 , wherein the nucleic acid encoding the RNA-guided endonuclease is mRNA.

16 . The method of claim 13 , wherein the nucleic acid encoding the RNA-guided endonuclease is DNA.

17 . The method of claim 16 , wherein the DNA is part of a vector that further comprises a sequence encoding the guide RNA.

18 . The method of claim 13 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a stem cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.

19 . The method of claim 13 , wherein the embryo is a non-human one cell animal embryo.

20 . A fusion protein comprising a CRISPR/Cas-like protein or fragment thereof and an effector domain.

21 . The fusion protein of claim 20 , wherein the CRISPR/Cas-like protein is derived from a Cas9 protein.

22 . The fusion protein of claim 21 , wherein the Cas9 protein is modified to lack at least one functional nuclease domain.

23 . The fusion protein of claim 21 , wherein the Cas9 protein is modified to lack all nuclease activity.

24 - 28 . (canceled)

29 . The fusion protein of claim 20 , wherein the effector domain is chosen from a transcriptional activation domain, a transcriptional repressor domain, and an epigenetic modification domain.

30 . The fusion protein of claim 20 , wherein the fusion protein further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, and a marker domain.

31 . (canceled)

32 . A method for modifying a chromosomal sequence or regulating expression of a chromosomal sequence in a cell or embryo, the method comprising introducing into the cell or embryo (a) at least one fusion protein or nucleic acid encoding at least one fusion protein, wherein the fusion protein comprises a CRISPR/Cas-like protein or a fragment thereof and an effector domain, and (b) at least one guide RNA or DNA encoding at least one guide RNA, wherein the one guide RNA guides the CRISPR/Cas-like protein of the fusion protein to a targeted site in the chromosomal sequence and the effector domain of the fusion protein modifies the chromosomal sequence or regulates expression of the chromosomal sequence.

33 . The method of claim 32 , wherein the CRISPR/Cas-like protein is derived from a Cas9 protein.

34 . The method of claim 33 , wherein the cas9 protein is modified to lack at least one functional nuclease domain.

35 . The method of claim 33 , wherein the cas9 protein is modified to lack all nuclease activity.

36 . The method of claim 32 , wherein fusion protein further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, and a marker domain.

37 . (canceled)

38 . The method of claim 32 , wherein one fusion protein or nucleic acid encoding one fusion protein and two guide RNAs or DNA encoding two guide RNAs are introduced into the cell or embryo.

39 . The method of claim 38 , wherein the fusion protein comprises a Cas9 protein or fragment thereof.

40 . The method of claim 32 , wherein two fusion proteins or nucleic acid encoding two fusion proteins and two guide RNAs or DNA encoding two guide RNAs are introduced into the cell or embryo.

41 . The method of claim 40 , wherein each fusion protein comprises a different Cas9 protein or fragment thereof.

42 - 43 . (canceled)

44 . The method of claim 32 , further comprising introducing into the cell or embryo at least one donor polynucleotide.

45 . The method of claim 32 , wherein the effector domain is chosen from an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.

46 . The method of claim 45 , wherein one fusion protein or nucleic acid encoding one fusion protein, and one guide RNA or DNA encoding one guide RNA are introduced into the cell or embryo.

47 . The method of claim 32 , wherein the cell is a human cell, a non-human mammalian cell, a stem cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.

48 . The method of claim 32 , wherein the embryo is a non-human one cell animal embryo.

Assignees

• Sigma Aldrich Co Llc

Inventors

• Chen Fuqiang
• Davis Gregory D
• Kang Qiaohua
• Knight Scott W

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/63

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

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/85 Primary

  for animal cells · CPC title