Crispr-cas component systems, methods and compositions for sequence manipulation

Patent metadata

Field	Value
Publication number	US-2016115489-A1
Application number	US-201614991083-A
Country	US
Kind code	A1
Filing date	Jan 8, 2016
Priority date	Dec 12, 2012
Publication date	Apr 28, 2016
Grant date	—

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 inducing cell death of one or more prokaryotic cell(s) comprising:
introducing one or more vectors into the prokaryotic cell(s); wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence; and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, are produced in the prokaryotic cell(s); wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within a target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence; wherein binding of the CRISPR complex to the target polynucleotide results in Cas9-directed cleavage at a targeted site in the prokaryote(s) and induces cell death.

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 prokaryote(s).

6 . The method of claim 1 wherein the prokaryote(s) is S. pneumoniae or E. coli.

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

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

• 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

• C12N2310/3519

  Fusion with another nucleic acid · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title