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

Abstract

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

1 . A gRNA comprising a nucleotide sequence selected from SEQ ID NOs: 149-284 or encoded by a polynucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 285-315, 321-323, and 326-329. 
     
     
         2 . A CRISPR/Cas-based base editing system comprising:
 the gRNA of  claim 1 ; and   a Cas protein or a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 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.   
     
     
         3 . The CRISPR/Cas-based base editing system of  claim 2 , wherein the fusion protein comprises a dCas9 domain and a transcriptional activator, or wherein the fusion protein comprises a dCas9 domain and a transcriptional repressor, or wherein the fusion protein comprises a dCas9 domain and a site-specific nuclease. 
     
     
         4 . The CRISPR/Cas-based base editing system of  claim 2 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 2 or 3. 
     
     
         5 . An isolated polynucleotide encoding the gRNA of  claim 1 . 
     
     
         6 . A vector comprising the isolated polynucleotide of  claim 5 . 
     
     
         7 . A cell comprising the isolated polynucleotide of  claim 5 . 
     
     
         8 . A kit comprising the isolated polynucleotide of  claim 5 . 
     
     
         9 . A method of site specific DNA cleavage in a target cell or a subject, the method comprising contacting the cell or the subject with an effective amount of the gRNA of  claim 1  and a fusion protein or Cas9 protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 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. 
     
     
         10 . A method of modulating gene expression in a cell, the method comprising contacting the cell with an effective amount of the gRNA of  claim 1  and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 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. 
     
     
         11 . The method of  claim 10 , wherein the gene expression of at least one target gene is modulated when gene expression levels of the at least one target gene are increased or decreased compared to normal gene expression levels for the at least one target gene. 
     
     
         12 . The method of  claim 10 , wherein the fusion protein comprises a dCas9 domain and a transcriptional activator, or a dCas9 domain and a transcriptional repressor, or a dCas9 domain and a site-specific nuclease. 
     
     
         13 . The method of  claim 12 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3. 
     
     
         14 . The method of  claim 10 , wherein the gRNA is encoded by a polynucleotide sequence and packaged into a lentiviral vector. 
     
     
         15 . The method of  claim 14 , wherein the lentiviral vector comprises an expression cassette comprising a promoter operably linked to the polynucleotide sequence encoding the gRNA. 
     
     
         16 . The method of  claim 15 , wherein the lentiviral vector further comprises a polynucleotide sequence encoding the fusion protein. 
     
     
         17 . 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 gRNA of  claim 1  and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 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. 
     
     
         18 . A method of genome editing in a cell, the method comprising administering to the cell an effective amount of the gRNA of  claim 1  and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 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. 
     
     
         19 . The method of  claim 19 , wherein the genome editing comprises deleting a mutant gene, rearranging a mutant gene, replacing a mutant gene, or inserting a transgene. 
     
     
         20 . A method of treating a subject, the method comprising:
 administering to the subject a DNA targeting system comprising the gRNA of  claim 1  and a fusion protein or Cas9 protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 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.