Engineered cas9 variants

Abstract

Certain embodiments are directed to modified or variant Cas9 proteins, and/or methods of using the same.

1 . A method for engineering a modified Cas9 protein, the method comprising modeling an active state of a Cas9 HNH domain by:
 (a) aligning a scissile phosphate and flanking nucleotides of a T4 Endo VII system (2QNC) to a corresponding tDNA stretch in a Cas9 pre-catalytic state complex (5F9R);   (b) calculating a tDNA transformation matrix from paired ββα-metal (ββα-Me) motifs in the two nucleases to produce a model of the Cas9-HNH domain docked at a cleavage site;   (c) repeating (a) and (b), replacing the Cas9 pre-catalytic state complex crystal structure (5F9R) with snapshot structures from sets of long conventional molecular dynamics (cMD) trajectories to obtain an optimized Cas9 complex;   (d) replacing an α segment of the ββα-Me motif in the optimized Cas9 complex from (c) with the corresponding part in a Mg 2+ -bound apo-Cas9 structure (4CMP); and   (e) performing long cMD simulations to obtain the active state of the Cas9 HNH domain.   
     
     
         2 . The method of  claim 1 , further comprising identifying amino acid residues in the active state of the Cas9 HNH domain model. 
     
     
         3 . The method of  claim 2 , wherein the identified amino acid residues are involved in non-specific DNA cleavage or off-target binding. 
     
     
         4 . The method of  claim 2 , further comprising modifying one or more of the identified amino acid residues to amino acids having a lower propensity for non-specific DNA cleavage or off-target binding. 
     
     
         5 . The method of  claim 4 , wherein the amino acids used for modification have lower hydrophobicity, lower positive charge, or higher polarizability than the corresponding wild-type residues. 
     
     
         6 . The method of  claim 4 , wherein the modified amino acid residues comprise at least three or four modifications to the amino acid sequence corresponding to SEQ ID NO:1, the modifications comprising one or more of N588A/R765A/N767A;
 N588A/Q695A/R765A/N767A; N588A/N692A/R765A/N767A; N588A/N692A/R765A/R925A;   N588A/N692A/N767A/R925A; N692A/R765A/N767A/R925A; Q695A/R765A/N767A/R925A;   N588A/N692A/R765A/K929A; N588A/N692A/N767A/K929A;   N692A/R765A/N767A/K929A; Q695A/R765A/N767A/K929A;   N497A/Q695A/R765A/N767A; K526A/K528A/N497A/Q926A; K526A/K528A/K929A;   K526A/R765A/N767A/Y1013A; K528A/R765A/N767A/Y1013A;   K526A/R765A/N767A/Q926A; N497A/K526A/R765A/N767A;   N497A/K528A/R765A/N767A; N497A/K526A/R765A/Q926A;   N497A/K528A/R765A/Q926A; N588A/R765A/N767A/S845D; N588A/R765A/N767A/R832A;   N588A/R765A/N767A/K862A; N588A/R765A/N767A/K866A; N588A/R765A/N767A/R859A;   N588A/R765A/N767A/Q844A; N588A/R765A/N767A/K810A;   N588A/R765A/N767A/K848A; N588A/R765A/N767A/E370A; N588A/R765A/N767A/E223A;   N497A/N692A/K1031A/S845D; N497A/N692A/K1031A/R832A;   N497A/N692A/K1031A/K862A; N497A/N692A/K1031A/K866A;   N497A/N692A/K1031A/R859A; N497A/N692A/K1031A/Q844A;   N497A/N692A/K1031A/K810A; N497A/N692A/K1031A/K848A;   N497A/N692A/K1031A/E370A; N497A/N692A/K1031A/E223A;   N497A/N695A/K1031A/S845D; N497A/N695A/K1031A/R832A;   N497A/N695A/K1031A/K862A; N497A/N695A/K1031A/K866A;   N497A/N695A/K1031A/R859A; N497A/N695A/K1031A/Q844A;   N497A/N695A/K1031A/K810A; N497A/N695A/K1031A/K848A;   N497A/N695A/K1031A/E370A; N497A/N695A/K1031A/E223A;   K526A/N695A/K1031A/S845D; K526A/N695A/K1031A/R832A;   K526A/N695A/K1031A/K862A; K526A/N695A/K1031A/K866A;   K526A/N695A/K1031A/R859A; K526A/N695A/K1031A/Q844A;   K526A/N695A/K1031A/K810A; K526A/N695A/K1031A/K848A;   K526A/N695A/K1031A/E370A; K526A/N695A/K1031A/E223A;   K528A/N695A/K1031A/S845D; K528A/N695A/K1031A/R832A;   K528A/N695A/K1031A/K862A; K528A/N695A/K1031A/K866A;   K528A/N695A/K1031A/R859A; K528A/N695A/K1031A/Q844A;   K528A/N695A/K1031A/K810A; K528A/N695A/K1031A/K848A;   K528A/N695A/K1031A/E370A; K528A/N695A/K1031A/E223A; N692A/R765A/Y1013A;   N692A/R765A/S845D/Y1013A; N692A/R765A/R832A/Y1013A;   N692A/R765A/K862A/Y1013A; N692A/R765A/K866A/Y1013A;   N692A/R765A/R859A/Y1013A; N692A/R765A/Q844A/Y1013A;   N692A/R765A/K810A/Y1013A; N692A/R765A/K848A/Y1013A;   N692A/R765A/E370A/Y1013A; N692A/R765A/E223A/Y1013A; N692A/R765A/Y1013A;   N692A/Q695A/K810A/Y1013A; N692A/Q695A/K848A/Y1013A; K526A/K528A/Y1013A;   K526A/K528A/K268A/Y1013A; R447A/K526A/K528A/Y1013A; R765A/K929A/H930A;   R765A/K929A/S845D/Y1013A; R765A/K929A/R832A/Y1013A;   R765A/K929A/K862A/Y1013A; R765A/K929A/K866A/Y1013A;   R765A/K929A/R859A/Y1013A; R765A/K929A/Q844A/Y1013A;   R765A/K929A/K810A/Y1013A; R765A/K929A/K848A/Y1013A;   R765A/K929A/E370A/Y1013A; R765A/K929A/E223A/Y1013A;   R765A/Q926A/K929A/H930A; R447A/K500A/R661A; K500A/N695A/K929A/S845D;   K500A/N695A/K929A/R832A; K500A/N695A/K929A/K862A;   K500A/N695A/K929A/K866A; K500A/N695A/K929A/R859A;   K500A/N695A/K929A/Q844A; K500A/N695A/K929A/K810A;   K500A/N695A/K929A/K848A; K500A/N695A/K929A/E370A; K500A/N695A/K929A/E223A;   R765A/R925/Q926A; R765A/R925/Q926/Y1013A; N14A/K961A/K968A;   N14A/K961A/K968A/S845D; N14A/K961A/K968A/K848A; R447A/R765A/Y1013A;   K526A/N588A/R765A/N767A; N588A/K929A/H930A/Y1013A; R447A/K526A/K929A;   N588A/N767A/Y1013A/K866A; N588A/N767A/Y1013A/S845D;   K268A/K526A/N588A/N767A; N14A/K526A/K866A/K1246A;   N14A/R447A/Y1013A/K1246A; N588A/R765A/D835A/K1246A;   N14A/R447A/R765A/S845D; K1244A/K1246A/K848A; K1244A/K1246A/K810A;   K1244A/K1246A/R832A; K1244A/K1246A/K862A; K1244A/K1246A/K866A;   K1244A/K1246A/R859A; K1244A/K1246A/E370A; K1244A/K1246A/E223A;   K1244A/K1246A/S845D; K1244A/K1246A/Q844A; K1244A/K1246A/Q844A/K1031A;   K1244A/K1246A/Q844A/Y1013A; K1244A/K1246A/Q844A/N695A;   K1244A/K1246A/Q844A/N692A; K1244A/K1246A/Q844A/N588A;   K1244A/K1246A/Q844A/N767A; K1244A/K1246A/Q844A/Q926A;   K268A/R447A/Y450A/K1031A; K268A/R447A/Y450A/Y1013A;   K268A/R447A/Y450A/N695A; K268A/R447A/Y450A/N692A;   K268A/R447A/Y450A/N588A; K268A/R447A/Y450A/N767A;   K268A/R447A/Y450A/Q926A; N14A/K268A/R447A/Y450A; N14A/Y450A/K526A/K528A;   N14A/Y450A/R765A/S845D; N14A/Y450A/R765A/R832A; N14A/Y450A/R765A/K862A;   N14A/Y450A/R765A/K866A; N14A/Y450A/R765A/R859A; N14A/Y450A/R765A/Q844A;   N14A/Y450A/R765A/K810A; N14A/Y450A/R765A/K848A; N14A/Y450A/R765A/E370A;   N14A/Y450A/R765A/E223A; R447A/Y450A/R765A/S845D; R447A/Y450A/R765A/R832A;   R447A/Y450A/R765A/K862A; R447A/Y450A/R765A/K866A; R447A/Y450A/R765A/R859A;   R447A/Y450A/R765A/Q844A; R447A/Y450A/R765A/K810A; R447A/Y450A/R765A/K848A;   R447A/Y450A/R765A/E370A; R447A/Y450A/R765A/E223A; K268A/R447A/R765A/S845D;   K268A/R447A/R765A/R832A; K268A/R447A/R765A/K862A; K268A/R447A/R765A/K866A;   K268A/R447A/R765A/R859A; K268A/R447A/R765A/Q844A; K268A/R447A/R765A/K810A;   K268A/R447A/R765A/K848A; K268A/R447A/R765A/E370A; K268A/R447A/R765A/E223A;   Q805A/D829A/N831A/D835A; R765A/D829A/D835A/Y1013A;   R918A/D829A/D835A/Y1013A; R895A/D829A/D835A/Y1013A;   K500A/D829A/D835A/Y1013A; K929A/D829A/D835A/Y1013A;   R780A/D829A/D835A/Y1013A; R783A/D829A/D835A/Y1013A;   R765A/D829A/D835A/N695A; R918A/D829A/D835A/N695A;   R895A/D829A/D835A/N695A; K500A/D829A/D835A/N695A;   K929A/D829A/D835A/N695A; R780A/D829A/D835A/N695A;   R783A/D829A/D835A/N695A; N695A/R780A/R783A/S845D; N695A/R780A/R783A/R832A;   N695A/R780A/R783A/K862A; N695A/R780A/R783A/K866A; N695A/R780A/R783A/R859A;   N695A/R780A/R783A/Q844A; N695A/R780A/R783A/K810A; N695A/R780A/R783A/K848A;   N695A/R780A/R783A/E370A; N695A/R780A/R783A/E223A; N692A/R780A/R783A/S845D;   N692A/R780A/R783A/R832A; N692A/R780A/R783A/K862A; N692A/R780A/R783A/K866A;   N692A/R780A/R783A/R859A; N692A/R780A/R783A/Q844A; N692A/R780A/R783A/K810A;   N692A/R780A/R783A/K848A; N692A/R780A/R783A/E370A; N692A/R780A/R783A/E223A;   N692A/R780A/N803A/S845D; N692A/R780A/N803A/R832A; N692A/R780A/N803A/K862A;   N692A/R780A/N803A/K866A, N692A/R780A/N803A/R859A; N692A/R780A/N803A/Q844A;   N692A/R780A/N803A/K810A; N692A/R780A/N803A/K848A; N692A/R780A/N803A/E370A;   N692A/R780A/N803A/E223A; N692A/R783A/N803A/S845D; N692A/R783A/N803A/R832A;   N692A/R783A/N803A/K862A; N692A/R783A/N803A/K866A; N692A/R783A/N803A/R859A;   N692A/R783A/N803A/Q844A; N692A/R783A/N803A/K810A;   N692A/R783A/N803A/K848A; N692A/R783A/N803A/E370A; N692A/R783A/N803A/E223A;   N695A/R783A/N803A/S845D; N695A/R783A/N803A/R832A; N695A/R783A/N803A/K862A;   N695A/R783A/N803A/K866A; N695A/R783A/N803A/R859A; N695A/R783A/N803A/Q844A;   N695A/R783A/N803A/K810A; N695A/R783A/N803A/K848A; N695A/R783A/N803A/E370A;   N695A/R783A/N803A/E223A; N695A/R783A/Y812A/S845D; N695A/R783A/Y812A/R832A;   N695A/R783A/Y812A/K862A; N695A/R783A/Y812A/K866A; N695A/R783A/Y812A/R859A;   N695A/R783A/Y812A/Q844A; N695A/R783A/Y812A/K810A;   N695A/R783A/Y812A/K848A; N695A/R783A/Y812A/E370A; N695A/R783A/Y812A/E223A;   K500A/N588A/S845D/Y1013A; K500A/N588A/R832A/Y1013A;   K500A/N588A/K862A/Y1013A; K500A/N588A/K866A/Y1013A;   K500A/N588A/R859A/Y1013A; K500A/N588A/Q844A/Y1013A;   K500A/N588A/K810A/Y1013A; K500A/N588A/K848A/Y1013A;   K500A/N588A/E370A/Y1013A; K500A/N588A/E223A/Y1013A;   K500A/N588A/S845D/Y1013A; N588A/N692A/K1244A/K1246A; R447A/R765A/N497A;   R447A/R765A/K929A; R447A/R765A/N767A; R447A/R765A/N767A/K558A;   R447A/R765A/N767A/R586A; R447A/R765A/N767A/K1244A;   R447A/R765A/N767A/K1246A; R447A/R765A/N767A; R447A/N695A/R765A/N767A;   R447A/R765A/N695A/K558A; R447A/R765A/N695A/R586A;   R447A/R765A/N695A/K1244A; R447A/R765A/N695A/K1246A;   R447A/R765A/N767A/K1246A; R447A/N695A/R765A/N767A;   R447A/R765A/N695A/K558A; R447A/R765A/N695A/R586A;   R447A/R765A/N695A/K1244A; R447A/R765A/N695A/K1246A;   R447A/N692A/R765A/N767A; R447A/R765A/N692/K558A; R447A/R765A/N692/R586A;   R447A/R765A/N692/K1244A; R447A/R765A/N692/K1246A; or N14A/R447A/R765A/S845A.   
     
     
         7 . The method of  claim 6 , wherein the modifications comprise:
 K526A/N588A/R765A/N767A; N588A/K929A/H930A/Y1013A; R447A/K526A/K929A;   N588A/N767A/Y1013A/K866A; N588A/N767A/Y1013A/S845D;   K268A/K526A/N588A/N767A; N14A/K526A/K866A/K1246A;   N14A/R447A/Y1013A/K1246A; N588A/R765A/D835A/K1246A; or   N14A/R447A/R765A/S845D.   
     
     
         8 . The method of  claim 6 , wherein the modifications comprise:
 N588A/R765A/D835A/K1246A or N14A/R447A/R765A/S845D.   
     
     
         9 . The method of  claim 6 , further comprising one or more modifications including modification of Asn14, Lys268, Glu370, Arg447, Tyr450, Asn497, Lys500, Lys526, Lys528, Lys558, Asn588, Arg661, Asn692, Gln695, Arg780, Arg783, Asn803, Gln805, Lys810, Tyr812, Asp829, Asn831, Arg832, Asp835, Gln844, Lys848, Lys862, Arg925, Gln926, Lys929, His930, Lys961, Lys968, Tyr1013, Lys1031, Lys1244, or Lys1246 corresponding to SEQ ID NO:1. 
     
     
         10 . The method of  claim 9 , wherein the modifications comprise substitution to an alanine, glycine, lysine, arginine, aspartic acid, or glutamic acid. 
     
     
         11 . The method of  claim 6 , wherein the modifications comprise at least four amino acid modifications. 
     
     
         12 . The method of  claim 1 , further comprising constructing 3D structure models of the engineered modified Cas9 proteins. 
     
     
         13 . The method of  claim 1 , further comprising evaluating the specificity of the engineered modified Cas9 proteins. 
     
     
         14 . The method of  claim 13 , wherein the specificity of the engineered modified Cas9 proteins is evaluated by comparing them with the specificity of a wild-type Cas9 protein or a Cas9 protein having other modifications. 
     
     
         15 . The method of  claim 13 , wherein the engineered modified Cas9 proteins have improved specificity compared to the specificity of the wild-type Cas9 protein. 
     
     
         16 . The method of  claim 15 , wherein the wild-type Cas9 protein is a  Streptococcus pyogenes  Cas9 protein. 
     
     
         17 . The method of  claim 13 , wherein the specificity is evaluated using a cleavage assay or a human cell-based enhanced GFP disruption assay. 
     
     
         18 . The method of  claim 1 , further comprising selecting one or more of the engineered modified Cas9 proteins for use in genome editing. 
     
     
         19 . A method for engineering a modified Cas9 protein having, the method comprising:
 modeling an active state of a Cas9 HNH domain by:
 (a) aligning a scissile phosphate and flanking nucleotides of a T4 Endo VII system (2QNC) to a corresponding tDNA stretch in a Cas9 pre-catalytic state complex (5F9R); 
 (b) calculating a tDNA transformation matrix from paired ββα-metal (ββα-Me) motifs in the two nucleases to produce a model of the Cas9-HNH domain docked at a cleavage site; 
 (c) repeating (a) and (b), replacing the Cas9 pre-catalytic state complex crystal structure (5F9R) with snapshot structures from sets of long conventional molecular dynamics (cMD) trajectories to obtain an optimized Cas9 complex; 
 (d) replacing an α segment of the ββα-Me motif in the optimized Cas9 complex from (c) with the corresponding part in a Mg 2+ -bound apo-Cas9 structure (4CMP); and 
 (e) performing long cMD simulations to obtain the active state of the Cas9 HNH domain; 
   identifying amino acid residues in the active state of the Cas9 HNH domain model involved in non-specific DNA cleavage or off-target binding;   engineering the modified Cas9 protein by modifying one or more of the identified amino acid residues to amino acids having lower propensity for non-specific DNA cleavage or off-target binding.