Engineered CRISPR-Cas9 Nucleases

Abstract

Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genomic engineering, epigenomic engineering, genome targeting, and genome editing.

1 .- 43 . (canceled) 
     
     
         44 . An isolated  Streptococcus pyogenes  Cas9 (SpCas9) protein that has at least 95% sequence identity to SEQ ID NO: 1, with mutations at all four of the following positions: Q695, Q926, N497, and R661, and wherein the SpCas9 further comprises mutations at one, two, or all three of L169, Y450, and D1135. 
     
     
         45 . The isolated protein of  claim 44 , wherein the protein retains the ability to interact with a guide RNA and target DNA. 
     
     
         46 . The isolated protein of  claim 44 , wherein the SpCas9 protein is fused to one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag. 
     
     
         47 . The isolated protein of  claim 44 , comprising the following mutations: N497A, R661A, Q695A, Q926A, and D1135E. 
     
     
         48 . The isolated protein of  claim 44 , comprising the following mutations: N497A, R661A, Q695A, Q926A, and L169A. 
     
     
         49 . The isolated protein of  claim 44 , comprising the following mutations: N497A, R661A, Q695A, Q926A, and Y450A. 
     
     
         50 . The isolated protein of  claim 44 , further comprising one or more mutations that decrease nuclease activity selected from the group consisting of mutations at D10, E762, D839, H983, or D986; and at H840 or N863. 
     
     
         51 . The isolated protein of  claim 50 , wherein the mutations that decrease nuclease activity are: (i) D10A or D10N, and (ii) H840A, H840N, or H840Y. 
     
     
         52 . A fusion protein comprising the isolated protein of  claim 44 , fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein. 
     
     
         53 . The fusion protein of  claim 52 , wherein the heterologous functional domain is a transcriptional activation domain. 
     
     
         54 . The fusion protein of  claim 53 , wherein the transcriptional activation domain is from VP64 or NFκB p65. 
     
     
         55 . The fusion protein of  claim 52 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain. 
     
     
         56 . The fusion protein of  claim 53 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID). 
     
     
         57 . The fusion protein of  claim 55 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1). 
     
     
         58 . The fusion protein of  claim 52 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA. 
     
     
         59 . The fusion protein of  claim 58 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein. 
     
     
         60 . The fusion protein of  claim 59 , wherein the TET protein is TET1. 
     
     
         61 . The fusion protein of  claim 52 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit. 
     
     
         62 . The fusion protein of  claim 61 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase. 
     
     
         63 . The fusion protein of  claim 52 , wherein the heterologous functional domain is a biological tether. 
     
     
         64 . The fusion protein of  claim 63 , wherein the biological tether is MS2, Csy4 or lambda N protein. 
     
     
         65 . The fusion protein of  claim 52 , wherein the heterologous functional domain is FokI. 
     
     
         66 . An isolated nucleic acid encoding the protein of  claim 44 . 
     
     
         67 . A vector comprising the isolated nucleic acid of  claim 66 . 
     
     
         68 . A host cell comprising the nucleic acid of  claim 66 . 
     
     
         69 . A method of altering the genome of a cell, the method comprising expressing in the cell or contacting the cell with the isolated protein of  claim 44 , linked to a guide RNA having a region complementary to a selected portion of the genome of the cell, whereby the genome of the cell is altered. 
     
     
         70 . The method of  claim 69 , wherein the isolated protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag. 
     
     
         71 . A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the isolated protein of  claim 44 , linked to a guide RNA having a region complementary to a selected portion of the dsDNA molecule, whereby the dsDNA molecule is altered.