Crispr-based genome modification and regulation

Patent metadata

Field	Value
Publication number	US-2017191082-A1
Application number	US-201715456204-A
Country	US
Kind code	A1
Filing date	Mar 10, 2017
Priority date	Dec 6, 2012
Publication date	Jul 6, 2017
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

What is claimed is:

1 . A method for modifying a chromosomal sequence in a eukaryotic cell by integrating a donor sequence, the method comprising introducing into the eukaryotic cell:
(i) a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II protein linked to at least one nuclear localization signal (NLS) or a nucleic acid encoding the CRISPR/Cas type II protein linked to at least one NLS, wherein the CRISPR/Cas type II protein is a Cas9 protein, and the nucleic acid encoding the CRISPR/Cas type II protein is codon optimized for expression in the eukaryotic cell; (ii) a guide RNA or DNA encoding the guide RNA, wherein the guide RNA comprises a first region that is complementary to a target site in the chromosomal sequence that is immediately followed by a protospacer adjacent motif, and a second region that interacts with the CRISPR/Cas type II protein; and (iii) a donor polynucleotide comprising the donor sequence;
wherein the guide RNA guides the CRISPR/Cas type II protein to the target site in the chromosomal sequence, the CRISPR/Cas type II protein introduces a double-stranded break at the target site, and repair of the double-stranded break by a DNA repair process leads to integration or exchange of the donor sequence into the chromosomal sequence.

2 . The method of claim 1 , wherein the CRISPR/Cas type II protein is linked to at least one fluorescent protein marker.

3 . The method of claim 1 , wherein the guide RNA is a single molecule.

4 . The method of claim 3 , wherein the guide RNA is enzymatically synthesized.

5 . The method of claim 1 , wherein the guide RNA comprises a crRNA and a tracrRNA.

6 . The method of claim 5 , wherein the crRNA is chemically synthesized and the tracrRNA is enzymatically synthesized, or both are enzymatically synthesized.

7 . The method of claim 1 , wherein DNA encoding the guide RNA is operably linked to a promoter sequence for expression in the eukaryotic cell and is part of a vector.

8 . The method of claim 1 , wherein the nucleic acid encoding the CRISPR/Cas type II protein is mRNA.

9 . The method of claim 1 , wherein the nucleic acid encoding the CRISPR/Cas type II protein is DNA.

10 . The method of claim 1 , wherein the nucleic acid encoding the CRISPR/Cas type II protein is part of a vector and the nucleic acid encoding the CRISPR/Cas type II protein is operably linked to a promoter sequence for expression in the eukaryotic cell.

11 . The method of claim 10 , wherein the vector further comprises sequence encoding the guide RNA that is operably linked to a promoter sequence for expression in the eukaryotic cell.

12 . The method of claim 1 , wherein the donor sequence comprises at least one nucleotide change relative to the target site in the chromosomal sequence.

13 . The method of claim 1 , wherein the donor sequence in the donor polynucleotide is flanked by sequences having substantial sequence identity to sequences on either side of the target site in the chromosomal sequence.

14 . The method of claim 1 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or single cell eukaryotic organism.

15 . A method for modifying a chromosomal sequence in a eukaryotic cell by integrating a donor sequence, the method comprising introducing into the eukaryotic cell:
(i) a nucleic acid encoding a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II protein linked to at least one nuclear localization signal (NLS), wherein the CRISPR/Cas type II protein is a Cas9 protein, and the nucleic acid encoding the CRISPR/Cas type II protein is codon optimized for expression in the eukaryotic cell; (ii) a guide RNA or DNA encoding the guide RNA, wherein the guide RNA comprises a first region that is complementary to a target site in the chromosomal sequence that is immediately followed by a protospacer adjacent motif, and a second region that interacts with the CRISPR/Cas type II protein; and (iii) a donor polynucleotide comprising the donor sequence;
wherein the nucleic acid encoding the CRISPR/Cas type II protein is RNA, the nucleic acid encoding the CRISPR/Cas type II protein is DNA, or the nucleic acid encoding the CRISPR/Cas type II protein is DNA that is part of a vector that further comprises DNA encoding the guide RNA; and
wherein the guide RNA guides the CRISPR/Cas type II protein to the target site in the chromosomal sequence, the CRISPR/Cas type II protein introduces a double-stranded break at the target site, and repair of the double-stranded break by a DNA repair process leads to integration or exchange of the donor sequence into the chromosomal sequence.

16 . The method of claim 15 , wherein the guide RNA is a single molecule.

17 . The method of claim 16 , wherein the guide RNA is enzymatically synthesized.

18 . The method of claim 15 , wherein the guide RNA comprises a crRNA and a tracrRNA.

19 . The method of claim 18 , wherein the crRNA is chemically synthesized and the tracrRNA is enzymatically synthesized, or both are enzymatically synthesized.

20 . The method of claim 15 , wherein the vector further comprises operably linked promoter control sequences.

21 . The method of claim 15 , wherein the donor sequence comprises at least one nucleotide change relative to the target site in the chromosomal sequence.

22 . The method of claim 15 , wherein the donor sequence in the donor polynucleotide is flanked by sequences having substantial sequence identity to sequences on either side of the target site in the chromosomal sequence.

23 . The method of claim 15 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or single cell eukaryotic organism.

Assignees

• Sigma Aldrich Co Llc

Inventors

• Chen Fuqiang
• Davis Gregory D

Classifications

• C12N15/63

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

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N2310/20

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

• C12N15/85

  for animal cells · CPC title

• C12N15/67

  General methods for enhancing the expression · CPC title