Crispr-cas systems, crystal structure and uses thereof

Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.

1 - 11 . (canceled) 
     
     
         12 . A method for reducing interaction of a  Streptococcus pyogenes  Cas9 (SpCas9) protein with a target DNA, comprising mutating one or more residues at Asn497, Trp659, Arg661, Gln695, Gln926, Glu1108, Leu169, Tyr450, Met495, Met694, or His698 of a corresponding wild-type SpCas9. 
     
     
         13 . A method for reducing interaction of a  Streptococcus pyogenes  Cas9 (SpCas9) protein with backbone phosphate groups of a target DNA, comprising mutating one or more residues at Asn497, Trp659, Arg661, Gln695, Gln926, or Glu1108 of a corresponding wild-type SpCas9. 
     
     
         14 . A method for reducing interaction of a  Streptococcus pyogenes  Cas9 (SpCas9) protein with C2′ atoms of a target DNA, comprising mutating one or more residues at Leu169, Tyr450, Met495, Met694, or His698 of a corresponding wild-type SpCas9. 
     
     
         15 . A composition comprising a  Streptococcus pyogenes  Cas9 (SpCas9) protein having reduced interaction with a target DNA, wherein the SpCas9 protein comprises one or more mutations at Asn497, Trp659, Arg661, Gln695, Gln926, Glu1108, Leu169, Tyr450, Met495, Met694, or His698 of a corresponding wild-type SpCas9. 
     
     
         16 . The composition of  claim 15 , wherein the SpCas9 protein comprises one or more mutations at Asn497, Trp659, Arg661, Gln695, Gln926, or Glu1108 which reduces interaction with backbone phosphate groups of the target DNA. 
     
     
         17 . The composition of  claim 15 , wherein the SpCas9 protein comprises one or more mutations at Leu169, Tyr450, Met495, Met694, or His698 which reduces interaction with C2′ atoms of the target DNA. 
     
     
         18 . The composition of  claim 15 , further comprising a CRISPR-Cas system guide RNA. 
     
     
         19 . The composition of  claim 18 , wherein the SpCas9 protein and the guide RNA forms a CRISPR-Cas complex.