Crispr-cas systems and methods for altering expression of gene products

Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. 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 utilizing the CRISPR-Cas system.

1 - 50 . (canceled) 
     
     
         51 . A method of selecting one or more prokaryotic cell(s), comprising:
 (a) introducing to a population of prokaryotic cells an engineered, programmable, non-naturally occurring CRISPR-Cas system comprising a Cas protein and one or more guide RNAs that target genomic loci of DNA molecules encoding one or more gene products in prokaryotic cell(s) of the population, wherein the one or more guide RNAs comprise a guide sequence, a tracr sequence, and a tracr mate sequence, and wherein the Cas protein and the one or more guide RNAs do not naturally occur together;   (b) allowing the Cas protein to cleave the genomic loci of the DNA molecules encoding the one or more gene products,   wherein cleavage of the genomic loci induces cell death, thereby allowing one or more prokaryotic cell(s) having one or more mutations in the genomic loci to be selected.   
     
     
         52 . The method of  claim 51 , wherein the Cas protein is a type II CRISPR system Cas protein. 
     
     
         53 . The method of  claim 52 , wherein the Cas protein is Cas9 enzyme. 
     
     
         54 . The method of  claim 53 , wherein the Cas9 enzyme is a  Streptococcus pyogenes  Cas9 enzyme. 
     
     
         55 . The method of  claim 51 , wherein the prokaryotic cells are  S. pneumoniae.    
     
     
         56 . The method of  claim 51 , wherein the one or more guide RNAs target an antibiotic resistance gene.