Using Truncated Guide RNAs (tru-gRNAs) to Increase Specificity for RNA-Guided Genome Editing

Abstract

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

1 .- 28 . (canceled) 
     
     
         29 . A complex comprising:
 a  Streptococcus pyogenes  Cas9 protein; and   a  Streptococcus pyogenes  gRNA molecule that includes a complementarity region at the 5′ end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target sequence, wherein the target sequence is immediately 5′ of a protospacer adjacent motif,   wherein the gRNA is a single gRNA, and   wherein in the presence of an  S. pyogenes  Cas9 protein, the gRNA complementary region binds and directs the Cas9 protein to the target sequence.   
     
     
         30 . The complex of  claim 29 , wherein the Cas9 protein is a catalytically active nuclease. 
     
     
         31 . The complex of  claim 29 , wherein the Cas9 protein is a catalytically active nickase. 
     
     
         32 . The complex of  claim 29 , wherein the Cas9 protein is a catalytically inactive Cas9 protein. 
     
     
         33 . The complex of  claim 29 , wherein the complementarity region of the gRNA consists of 17 nucleotides. 
     
     
         34 . The complex of  claim 29 , wherein the complementarity region of the gRNA consists of 18 nucleotides. 
     
     
         35 . A DNA molecule encoding the complex of  claim 29 . 
     
     
         36 . A vector comprising the DNA molecule of  claim 35 . 
     
     
         37 . A host cell expressing the vector of  claim 36 . 
     
     
         38 . A complex comprising:
 a  Streptococcus pyogenes  Cas9 protein; and   a  Streptococcus pyogenes  gRNA molecule that includes a complementarity region at the end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target sequence, wherein the target sequence is immediately 5′ of a protospacer adjacent motif,   wherein the gRNA is a CRISPR RNA (crRNA), and   wherein in the presence of an  S. pyogenes  Cas9 protein, the gRNA complementary region binds and directs the Cas9 protein to the target sequence.   
     
     
         39 . A vector comprising a DNA molecule encoding:
 a  Streptococcus pyogenes  gRNA molecule that includes a complementarity region at the end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target sequence, wherein the target sequence is immediately 5′ of a protospacer adjacent motif,
 wherein the gRNA is a single gRNA or a CRISPR RNA (crRNA), and 
 wherein in the presence of an  S. pyogenes  Cas9 nuclease, the gRNA complementary region binds and directs the Cas9 nuclease to the target sequence; and 
 a promoter. 
   
     
     
         40 . The vector of  claim 39 , wherein the promoter is a constitutive promoter. 
     
     
         41 . The vector of  claim 39 , wherein the promoter is an inducible promoter. 
     
     
         42 . The vector of  claim 39 , wherein the promoter is a weak promoter. 
     
     
         43 . The vector of  claim 39 , wherein the expression vector is a bacterial expression vector. 
     
     
         44 . The vector of  claim 39 , wherein the expression vector is a eukaryotic expression vector. 
     
     
         45 . The vector of  claim 39 , wherein the promoter is an RNA Pol III promoter. 
     
     
         46 . The vector of  claim 39 , wherein the promoter is selected from an H1, U6, or 7SK promoter. 
     
     
         47 . The vector of  claim 39 , wherein the promoter is a T7 promoter. 
     
     
         48 . A host cell expressing the vector of  claim 47 .