Guide rna with chemical modifications

Abstract

The present invention relates to modified guide RNAs and their use in clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems.

We claim: 
     
         1 . A synthetic guide RNA comprising:
 (c) a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target sequence in a polynucleotide, (ii) a stem sequence; and   (d) a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence,   
       wherein the synthetic guide RNA comprises one or more modifications, and wherein the synthetic guide RNA has gRNA functionality. 
     
     
         2 . The synthetic guide RNA of  claim 1 , wherein one or more of the modifications comprises a stability-enhancing modification. 
     
     
         3 . The synthetic guide RNA of  claim 2 , wherein one or more of the stability-enhancing modifications is located in the guide sequence. 
     
     
         4 . The synthetic guide RNA of  claim 1 , comprising less than 26 consecutive 2′-O-methyl modified nucleotides at a 5′ end of the guide RNA. 
     
     
         5 . The synthetic guide RNA of  claim 1 , comprising a Z base replacing a cytosine in the synthetic guide RNA. 
     
     
         6 . The synthetic guide RNA of  claim 1 , comprising at least one 2-thiouracil at a position corresponding to a uridine that can engage in U-G wobble pairing with a potential off-target sequence. 
     
     
         7 . The synthetic guide RNA of  claim 1 , comprising one or more modifications selected from the group consisting of a 2′-O-methyl nucleotide with a 3′-phosphorothioate group, a 2′-O-methyl nucleotide with a 3′-phosphonoacetate group, a 2′-O-methyl nucleotide with a 3′-thiophosphonoacetate group, and a 2′-deoxy nucleotide with a 3′-phosphonoacetate group. 
     
     
         8 . The synthetic guide RNA of  claim 1 , comprising at least two modifications. 
     
     
         9 . The synthetic guide RNA of  claim 1 , comprising up to 50 modifications. 
     
     
         10 . The synthetic guide RNA of  claim 1 , comprising a single RNA strand or two separate RNA strands, and one or more modifications at a 5′ end of each RNA strand, at a 3′ end of each RNA strand, or at both a 5′ end and a 3′ end of each RNA strand. 
     
     
         11 . The synthetic guide RNA of  claim 1 , wherein one or more of the modifications alters base-pairing thermostability. 
     
     
         12 . The synthetic guide RNA of  claim 11 , wherein said one or more modifications enhances the base-pairing thermostability. 
     
     
         13 . The synthetic guide RNA of  claim 11 , wherein said one or more modifications decreases the base-pairing thermostability. 
     
     
         14 . The synthetic guide RNA of  claim 1 , comprising one or more 2′-O-methylA-2′-O-methylU base pairs. 
     
     
         15 . The synthetic guide RNA of  claim 1 , wherein one or more of the modifications is a specificity-altering modification. 
     
     
         16 . A method for genomic editing to modify a DNA sequence, or regulating the expression of a gene of interest, or cleaving a target polynucleotide comprising: contacting the DNA sequence, the gene of interest, or the target polynucleotide with a CRISPR-associated protein and the synthetic guide RNA of any of the foregoing claims, and editing, regulating or cleaving the DNA sequence, the gene of interest or the target polynucleotide. 
     
     
         17 . The method of  claim 16 , wherein the method is performed in vitro, and the synthetic guide RNA comprises 15 or more 2′-O-methyl modifications throughout the guide sequence. 
     
     
         18 . A set or library of RNA molecules comprising two or more synthetic guide RNAs of  claim 1 . 
     
     
         19 . A kit comprising the synthetic guide RNA of  claim 1 . 
     
     
         20 . An array of RNA molecules comprising two or more synthetic guide RNAs of  claim 1 .