CRISPR-CAS COMPONENT SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION
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.
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.