Cas9-cas9 fusion proteins

Abstract

The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

1 - 42 . (canceled) 
     
     
         43 . A method for genome editing of DNA within a cell, comprising
 i) delivering a fusion protein comprising a first Cas9 nuclease, said first nuclease comprising a protospacer adjacent motif recognition domain having a lysine-substituted, alanine-substituted or serine-substituted arginine residue and a second Cas9 nuclease;   ii) delivering a first guide RNA for the first Cas9 comprising a guide sequence element that is complementary to a first target site,   iii) delivering a second guide RNA for the second Cas9 comprising a guide sequence element that is complementary to a second target site; and   iv) cleaving the first target site with the first Cas9 nuclease.   
     
     
         44 . The method of  claim 43 , wherein the second Cas9 nuclease is an orthogonal Cas9 isoform. 
     
     
         45 . The method of  claim 44 , wherein the first target site comprises a sequence complementary to the guide sequence element of the first guide RNA and a protospacer adjacent motif sequence for the first Cas9 nuclease, and the second target site comprises a sequence complementary to the guide sequence element of the second guide RNA and a protospacer adjacent motif sequence for the second Cas9 nuclease. 
     
     
         46 . The method of  claim 43 , further comprising cleaving the second target site with the second Cas9 nuclease. 
     
     
         47 . The method of  claim 46 , wherein a genomic deletion is generated. 
     
     
         48 . The method of  claim 43 , wherein said first and second Cas9 nucleases are selected from the group consisting of  Streptococcus pyogenes  Cas9 (SpCas9),  Staphylococcus aureus  Cas9 (SaCas9),  Neisseria meningitidis  Cas9 (NmCas9) and  Actinomyces naeslundii  Cas9 (AnCas9). 
     
     
         49 . The method of  claim 43 , wherein said substituted arginine protospacer adjacent motif (PAM) recognition residue participates in a base-specific binding or contacts a phosphodiester backbone. 
     
     
         50 . The method of  claim 43 , wherein said substituted arginine PAM recognition residue participates in a base-specific binding. 
     
     
         51 . The method of  claim 43 , wherein said substituted arginine PAM recognition residue contacts a phosphodiester backbone residue. 
     
     
         52 . The method of  claim 43 , wherein the first Cas9 nuclease has a single point mutation. 
     
     
         53 . The method of  claim 43 , wherein the first Cas9 nuclease has a double mutation. 
     
     
         54 . The method of  claim 43 , wherein said first Cas9 nuclease is selected from the group consisting of SpCas9.sup.R1333K, SpCas9.sup.R1333S, SpCas9R.sup.1335K, NmCas9.sup.R1025A and NmCas9.sup.K1013A/R1025A. 
     
     
         55 . The method of  claim 43 , wherein said second Cas9 nuclease is selected from the group consisting of a Cas9 nickase and a nuclease-dead Cas9 (dCas9). 
     
     
         56 . The method of  claim 43   1 , wherein said second Cas9 nuclease is selected from the group consisting of nuclease-dead NmCas9 (NmdCas9), NmCas9 nuclease, NmCas9 HNH nickase, and NmCas9 RuvC nickase. 
     
     
         57 . The method of  claim 43 , wherein said first and second Cas9 nucleases are bound to a guide RNA comprising a guide sequence element. 
     
     
         58 . The method of  claim 56 , wherein said guide sequence element is truncated. 
     
     
         59 . The method of  claim 43 , wherein the first Cas9 nuclease is selected from the group consisting of SpCas9.sup.R1333K, SpCas9.sup.R1333S, and SpCas9R.sup.1335K, and wherein the second Cas9 nuclease is NmCas9 nuclease. 
     
     
         60 . The method of  claim 43 , wherein the fusion protein comprises a linker. 
     
     
         61 . The method of  claim 60 , wherein the fusion protein comprises a linker that ranges between 20 and sixty amino acids. 
     
     
         62 . The method of  claim 60 , wherein the first guide RNA is a single guide RNA or a combination of crRNA and tracrRNA, and wherein the second guide RNA is a single guide RNA or a combination of crRNA and tracrRNA.