High Specificity Genome Editing Using Chemically Modified Guide RNAs

Abstract

The present invention relates to guide RNAs having chemical modifications and their use in CRISPR-Cas systems. The chemically modified guide RNAs have enhanced specificity for target polynucleotide sequences. The present invention also relates to methods of using chemically modified guide RNAs for cleaving or nicking polynucleotides, and for high specificity genome editing.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . The synthetic guide RNA of  claim 13 , wherein said at least one modification is selected from a phosphonoacetate internucleotide linkage (P), a thiophosphonoacetate internucleotide linkage (SP), and a 2′-modification that confers a C3′-endo sugar pucker configuration or a combination thereof. 
     
     
         4 . The synthetic guide RNA of  claim 3 , wherein said 2′-modification is selected from 2′-O-methyl, 2′-fluoro, and 2′-O-(2-methoxyethyl). 
     
     
         5 . The synthetic guide RNA of  claim 13 , wherein said at least one modification is selected from a 2′-deoxy-3′-phosphonoacetate (DP), 2′-O-deoxy-3′-thiophosphonoacetate (DSP), 2′-O-methyl-3′-phosphonoacetate (MP) and 2′-O-methyl-3′-thiophosphonoacetate (MSP). 
     
     
         6 . The synthetic guide RNA of  claim 5 , wherein said at least one modification is located at position 5-N or 11-N of the guide sequence, or a combination thereof. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . A synthetic guide RNA comprising:
 (a) a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, wherein the target polynucleotide comprises a target sequence adjacent to a PAM sequence, (ii) a stem sequence; and   (b) a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence,   wherein the guide sequence consists of 20-N nucleotides, where N is an integer between −10 and 6;   
       wherein the guide sequence further comprises at least one phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at any position from position 4-N to 20-N of the guide sequence, and wherein said modification is not at position 15-N of the guide sequence. 
     
     
         14 . The synthetic guide RNA of  claim 13 , wherein said phosphonocarboxylate internucleotide linkage is selected from a phosphonoacetate linkage (P) and a thiophosphonoacetate linkage (SP). 
     
     
         15 . The synthetic guide RNA of  claim 13 , further comprising at least one modification at the 5′-end, 3′-end or both ends of said guide RNA. 
     
     
         16 . The synthetic guide RNA of  claim 13 , wherein said guide RNA is a single guide RNA. 
     
     
         17 . The synthetic guide RNA of  claim 15 , wherein said at least one modification at the 5′-end, the 3′-end, or both ends is independently selected from a 2′-O-methyl (M), a phosphorothioate internucleotide linkage (S), a phosphonoacetate internucleotide linkage (P), a thiophosphonoacetate internucleotide linkage (SP), a 2′-O-methyl-3′-phosphoroatioate (MS), a 2′-O-methyl-3′-phosphosphonoacetate (MP) and a 2′-O-methyl-3′-thiophosphosphonoacetate (MSP), a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-O-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . A method for enhancing the specificity of a CRISPR function, comprising:
 selecting a target polynucleotide;   providing at least one synthetic guide RNA of  claim 13 ;   forming a gRNA:Cas protein complex comprising a Cas protein and the synthetic guide RNA; and   contacting the target polynucleotide with the gRNA:Cas protein complex;   wherein said Cas protein is provided as a protein or as a polynucleotide encoding said Cas protein.   
     
     
         21 . The method of  claim 20 , wherein said guide RNA is a single guide RNA. 
     
     
         22 . The method of  claim 20 , wherein said guide RNA further comprises at least one modification at the 5′-end, the 3′-end, or both ends of said guide RNA. 
     
     
         23 . The method of  claim 22 , wherein said at least one modification at the 5′-end, the 3′-end, or both ends is independently selected from a 2′-O-methyl (M), a phosphorothioate internucleotide linkage (S), a phosphonoacetate internucleotide linkage (P), a thiophosphonoacetate internucleotide linkage (SP), a 2′-O-methyl-3′-phosphoroatioate (MS), a 2′-O-methyl-3′-phosphosphonoacetate (MP) and a 2′-O-methyl-3′-thiophosphosphonoacetate (MSP), a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-O-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof. 
     
     
         24 . The method of  claim 20 , wherein said guide RNA comprises at least one modification selected from a phosphonoacetate internucleotide linkage and a thiophosphonoacetate internucleotide linkage or a combination thereof. 
     
     
         25 . The method of  claim 20 , wherein said guide RNA comprises at least one modification selected from a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-O-deoxy-3′-thiophosphonoacetate (DSP), 2′-O-methyl-3′-phosphonoacetate (MP) and 2′-O-methyl-3′-thiophosphonoacetate (MSP). 
     
     
         26 . The method of  claim 20 , wherein said contacting of said polynucleotide target with said gRNA:Cas protein complex is performed in a cell and wherein said forming said complex is performed outside or inside the cell. 
     
     
         27 . The method of  claim 26 , wherein said Cas protein is a Cas9 protein. 
     
     
         28 . The method of  claim 27 , wherein said target polynucleotide is located within the HBB gene, the IL2RG gene, or the VEGFA gene. 
     
     
         29 . The method of  claim 28 , wherein the target polynucleotide comprises GCCCCACAGGGCAGTAA (SEQ ID NO: 8) of the HBB gene, TAATGATGGCTTCAACA (SEQ ID NO: 10) of the IL2RG gene, or GAGTGAGTGTGTGCGTG (SEQ ID NO: 192) of the VEGFA gene. 
     
     
         30 . The method of  claim 20 , wherein said forming is performed outside of a cell.