Methods and compositions for nuclease design

Patent metadata

Field	Value
Publication number	US-9970001-B2
Application number	US-201514731821-A
Country	US
Kind code	B2
Filing date	Jun 5, 2015
Priority date	Jun 5, 2014
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

Methods and compositions for genetic alteration of cells are provided.

First claim

What is claimed is:

1. A Cas polypeptide comprising a S. pyogenes Cas 9 (spCas9) protein, wherein the Cas polypeptide that forms a complex with a single guide RNA (sgRNA) that binds to a target sequence in an endogenous gene, the target sequence comprising a protospacer adjacent motif (PAM) sequence of 5′VGGC (i.e. 5′ AGGC, CGGC or GGGC) at the 3′ end of the target sequence, wherein the complex exhibits increased binding specificity to and cleavage of the target sequence by the Cas/sgRNA complex as compared to a target sequence comprising a PAM sequence of NGGD (i.e. 5′ NGGA, NGGG or NGGT).

2. The Cas polypeptide of claim 1 , wherein the spCas9 polypeptide is a mutant spCas9 protein and the mutations are in the α-helical lobe of the Cas9 protein.

3. The Cas polypeptide of claim 2 , wherein the mutations are in the region of the Cas9 protein extending between amino acid 78 to amino acid 718 of SEQ ID NO:2.

4. The Cas polypeptide of claim 2 , wherein the mutations are in the C-terminal domain of the Cas polypeptide.

5. The Cas polypeptide of claim 4 , wherein the mutations are between amino acids 1099 to 1368 or 1200 to 1368 of SEQ ID NO:2.

6. The Cas polypeptide of claim 2 , wherein the mutation is in an altered tryptophan containing loop between amino acids 447-502 or 1102-1137 of SEQ ID NO:2.

7. A complex comprising the Cas polypeptide associated with the sgRNA of claim 1 and the target sequence in the endogenous gene.

8. The complex of claim 7 , wherein the target sequence is in an endogenous safe harbor gene.

9. A method of cleaving a target sequence in a cell, the method comprising generating a complex of claim 7 into the cell such that the target is cleaved.

10. A cell comprising a cleaved target made by the method of claim 9 .

Assignees

• Sangamo Biosciences Inc
• Sangamo Therapeutics Inc

Inventors

• Miller Jeffrey C

Classifications

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/63

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

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title