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 chemically-synthesized single molecule CRISPR guide RNA (gRNA) comprising:
 a 5′-end and a 3′-end;   one or more chemically modified nucleotides within five nucleotides from said 5′-end or said 3′-end, or within five nucleotides from each of said 5′-end and said 3′-end, wherein each of the one or more chemically modified nucleotides is independently selected from 2′-O-methyl-3′-phosphorothioate (MS), 2′-O-methyl-3′-phosphonoacetate (MP), and 2′-O-methyl-3′-thiophosphonoacetate (MSP);   a guide sequence that is 18-22 nucleotides long and is capable of hybridizing to a target sequence in a target polynucleotide; and   a scaffold region that can interact with a Cas9 protein to form a gRNA:Cas9 complex, wherein said complex is capable of cleaving both strands of the target polynucleotide when the Cas9 protein has double-strand nuclease activity.   
     
     
         2 . The gRNA of  claim 1 , wherein the gRNA does not comprise 26 or more consecutive chemically modified nucleotides from the 5′-end that consist of 2′-O-methyl. 
     
     
         3 . The gRNA of  claim 2 , wherein the gRNA does not comprise 26 or more consecutive chemically modified nucleotides from the 5′-end that consist of 2′-deoxy. 
     
     
         4 . The gRNA of  claim 3 , wherein the Cas9 protein is from  Streptococcus pyogenes.    
     
     
         5 . The gRNA of  claim 3 , wherein the guide sequence is 20 nucleotides long. 
     
     
         6 . The gRNA of  claim 3 , wherein said one or more chemically modified nucleotides comprises two consecutive modified nucleotides independently selected from MS, MP, and MSP. 
     
     
         7 . The gRNA of  claim 3 , wherein said one or more chemically modified nucleotides consists of two consecutive modified nucleotides independently selected from MS, MP, and MSP. 
     
     
         8 . The gRNA of  claim 3 , wherein said one or more chemically modified nucleotides comprises two consecutive MS modified nucleotides or two consecutive MP or MSP modified nucleotides. 
     
     
         9 . The gRNA of  claim 3 , wherein said one or more chemically modified nucleotides consists of two consecutive MS modified nucleotides or two consecutive MP or MSP modified nucleotides. 
     
     
         10 . The gRNA of  claim 3 , wherein said one or more chemically modified nucleotides comprises three consecutive MS modified nucleotides or three consecutive MP or MSP modified nucleotides. 
     
     
         11 . The gRNA of  claim 3 , wherein said one or more chemically modified nucleotides consists of three consecutive MS modified nucleotides or three consecutive MP or MSP modified nucleotides. 
     
     
         12 . The gRNA of  claim 3 , wherein said one or more chemically modified nucleotides comprises at least two modified nucleotides independently selected from MS, MP and MSP within five nucleotides from each of the 5′-end and the 3′-end. 
     
     
         13 . The gRNA of  claim 3  wherein said one or more chemically modified nucleotides consists of two modified nucleotides independently selected from MS, MP and MSP within five nucleotides from each of the 5′-end and the 3′-end. 
     
     
         14 . The gRNA of  claim 3  wherein said chemically modified nucleotides within five nucleotides from the 5′-end or the 3′-end, or within five nucleotides from each of said 5′-end and said 3′-end, consist of two to five modified nucleotides independently selected from MS, MP and MSP. 
     
     
         15 . The gRNA of  claim 1  wherein said one or more chemically modified nucleotides is located within 3 nucleotides from each of the 5′ and the 3′ ends. 
     
     
         16 . A ribonucleoprotein (RNP) comprising the gRNA of  claim 1  and a Cas protein. 
     
     
         17 . A method of editing, regulating gene expression of, detecting, or cleaving a target polynucleotide, the method comprising:
 providing a gRNA of  claim 1  and a Cas protein;   contacting the target polynucleotide with the guide RNA, thereby editing, regulating gene expression of, detecting, or cleaving the target polynucleotide.   
     
     
         18 . The method of  claim 17 , wherein the Cas protein has double-strand nuclease activity. 
     
     
         19 . The method of  claim 17 , wherein the Cas protein has a single-strand nicking activity. 
     
     
         20 . The method of  claim 17 , wherein the Cas protein lacks nuclease activity. 
     
     
         21 . The method of  claim 17 , wherein the Cas protein is a fusion protein. 
     
     
         22 . The method of  claim 17 , wherein the contacting occurs in a cell. 
     
     
         23 . The method of  claim 17 , wherein the Cas protein is provided to a cell as an mRNA encoding the Cas protein. 
     
     
         24 . The method of  claim 23 , wherein the mRNA is provided to the cell along with the gRNA. 
     
     
         25 . The method of  claim 17 , wherein the Cas protein is provided to a cell as a protein. 
     
     
         26 . The method of  claim 25 , wherein the Cas protein is provided to the cell along with the gRNA in a ribonucleoprotein. 
     
     
         27 . The method of  claim 17 , wherein the gRNA comprises at least two consecutive modified nucleotides independently selected from MS, MP and MSP within five nucleotides from each of the 5′- and the 3′-end. 
     
     
         28 . The method of  claim 27 , wherein the guide sequence is 20 nucleotides long. 
     
     
         29 . A set or a library of gRNAs comprising at least one gRNA of  claim 1 . 
     
     
         30 . A kit comprising at least one gRNA of  claim 1  and at least one Cas protein and/or at least one mRNA encoding a Cas protein.