Compositions and methods of improving specificity in genomic engineering using rna-guided endonucleases

Abstract

Disclosed herein are optimized guide RNAs (gRNAs) and methods of designing and using said optimized gRNAs that have increased target binding specificity and reduced off-target binding.

What is claimed: 
     
         1 - 68 . (canceled) 
     
     
         69 . A method for generating an optimized gRNA that has increased target specificity, the method comprising:
 a) generating a polynucleotide sequence of a full-length gRNA comprising a protospacer-targeting sequence, wherein:
 the protospacer-targeting sequence is complementary to a protospacer sequence of a target region in a target gene; and 
 the full-length gRNA is known or predicted to have off-target activity to at least one off-target site; 
   b) generating a plurality of hairpin gRNAs (hp-gRNAs) of different sequences, wherein each hp-gRNA comprises a polynucleotide sequence of the full-length gRNA, a polynucleotide segment at the 5′ end or the 3′ end of the full-length gRNA and optionally a linker between the gRNA and the polynucleotide segment, wherein the polynucleotide segment is complementary to at least a portion of the protospacer-targeting region; and   c) identifying a hp-gRNA from the plurality of hp-gRNAs that has increased target binding specificity compared to the full-length gRNA.   
     
     
         70 . The method of  claim 69 , wherein the polynucleotide segment of each of the plurality of hp-gRNAs has a length of between 2 and 20 nucleotides. 
     
     
         71 . The method of  claim 69 , wherein the linker is between 1 and 20 nucleotides in length. 
     
     
         72 . The method of  claim 69 , wherein the identified hp-gRNA has a decreased binding to the at least one off-target site. 
     
     
         73 . The method of  claim 69 , wherein identifying the hp-gRNA that has increased target binding specificity comprises calculating the invasion kinetics and lifetime of the full-length gRNA and hp-gRNA of the plurality remaining invaded in the protospacer sequence and the least one off-target site. 
     
     
         74 . The method of  claim 69 , wherein the identified hp-gRNA has a binding lifetime greater than or equal to that of the full-length gRNA at the protospacer sequence, and/or a binding lifetime less than or equal to that of the full-length gRNA at the at least one off-target site. 
     
     
         75 . The method of  claim 69 , wherein the identified hp-gRNA has a binding lifetime greater than or equal to that of the full-length gRNA at the protospacer sequence, and a binding lifetime less than or equal to that of the full-length gRNA for at least the top 3 off-target sites. 
     
     
         76 . The method of  claim 69 , wherein the polynucleotide segment comprises one or more wobble base pairs. 
     
     
         77 . The method of  claim 69 , wherein the optimized gRNA is for use in a CRISPR/Cas system. 
     
     
         78 . The method of  claim 77 , wherein the CRISPR/Cas system is a CRISRP/Cas9-based system. 
     
     
         79 . The method of  claim 78 , wherein the polynucleotide segment is at the 5′ end of the full-length gRNA. 
     
     
         80 . The method of  claim 77 , wherein the CRISPR/Cas system is a CRISRP/Cpf1-based system. 
     
     
         81 . The method of  claim 80 , wherein the polypeptide segment is at the 3′ end of the full-length gRNA. 
     
     
         82 . A hairpin gRNA (hp-RNA) identified by the method of  claim 69 . 
     
     
         83 . A hairpin gRNA (hp-gRNA) comprising:
 (i) a gRNA comprising a protospacer-targeting sequence of a target region of between about 15 and 20 nucleotides;   (ii) a polynucleotide segment at the 5′ end or the 3′ end of the gRNA, wherein the polynucleotide segment is complementary to a portion of the protospacer-targeting region; and   (iii) optionally a linker between the gRNA and the polynucleotide segment.   
     
     
         84 . A composition comprising the hp-gRNA of  claim 82  and a CRISPR/Cas system. 
     
     
         85 . The composition of  claim 84 , wherein the CRISRP/Cas system is a CRISRP/Cas9-based system comprising an inactivated Cas9 (dCas9) protein. 
     
     
         86 . A vector comprising a polynucleotide encoding a CRISPR/Cas system and the hp-gRNA of  claim 82 . 
     
     
         87 . A method of epigenomic editing in a target cell or a subject, the method comprising contacting a cell or a subject with an effective amount of the hp-gRNA of  claim 82  and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas protein and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity. 
     
     
         88 . A method of modulating gene expression in a cell, the method comprising contacting the cell with an effective amount of the hp-gRNA of  claim 82  and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas protein and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity. 
     
     
         89 . A method of genome editing in a cell, the method comprising contacting the cell with an effective amount of the hp-gRNA of  claim 82  and a Cas protein.