CRISPR-Cas component systems, methods and compositions for sequence manipulation

Patent metadata

Field	Value
Publication number	US-9822372-B2
Application number	US-201614990444-A
Country	US
Kind code	B2
Filing date	Jan 7, 2016
Priority date	Dec 12, 2012
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

First claim

What is claimed is:

1. A method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in one or more prokaryotic cell(s), the method comprising:
introducing one or more vectors into the prokaryotic cell(s);
wherein the one or more vectors drive expression of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template for recombination into a targeted chromosomal locus comprising a target polynucleotide;
and all of the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and a tracr sequence, and the editing template are produced in the prokaryotic cell(s), whereby a CRISPR complex forms; the CRISPR complex comprising the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence;
wherein the CRISPR complex binds to the target polynucleotide;
wherein the editing template is introduced into the target polynucleotide through recombination, wherein the editing template comprises one or more mutations of the target polynucleotide that alter either a protospacer-adjacent motif (PAM) sequence or a protospacer sequence, and that abolishes CRISPR enzyme cleavage of the target polynucleotide;
wherein in those cells that have not had the editing template introduced there is cleavage of the target polynucleotide by the CRISPR complex and cell death; and
selecting one or more prokaryotic cell(s) in which the one or more mutations have been introduced.

2. The method of claim 1 , wherein the CRISPR enzyme is a type II CRISPR system enzyme.

3. The method of claim 1 , wherein the CRISPR enzyme is a Cas9.

4. The method of claim 3 , wherein the Cas9 is S. pyogenes Cas9.

5. The method of claim 1 wherein the CRISPR enzyme is not endogenous to the prokaryotic cell(s).

6. The method of claim 1 wherein the prokaryotic cell(s) are S. pneumoniae or E. coli.

7. The method of claim 1 wherein the one or more vectors are plasmids.

8. The method of claim 1 wherein the one or more mutations alter the PAM sequence of the target polynucleotide.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Univ Rockefeller

Inventors

• Zhang Feng
• Cox David Benjamin Turitz
• Marraffini Luciano
• Bikard David Olivier
• Jiang Wenyan
• Sanjana Neville Espi

Classifications

• C12N15/63

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

• C12N9/22 Primary

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

• C12N15/8509

  for producing genetically modified animals, e.g. transgenic · CPC title

• C12N15/1082

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title

• C12N2310/3519

  Fusion with another nucleic acid · CPC title