High specificity genome editing using chemically modified guide RNAs
AssigneeAgilent Technologies, Inc.
InventorsDouglas J Dellinger, Daniel E Ryan, Subhadeep Roy, Jeffrey R Sampson
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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.
Claims23
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 site, and (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 3;
- and wherein the guide sequence comprises at least one modification located at position 4-N, 5-N, 7-N, 9-N, 10-N or 11-N relative to the 5′-end of the guide sequence, or a combination thereof;
- wherein the synthetic guide RNA has enhanced specificity for the target sequence compared to a guide RNA without said modification, and wherein said at least one modification is 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-(2-methoxyethyl) (MOE), a 2′-O-(2-methoxyethyl)-3′-phosphorothioate (MOES), a 2′-O-(2-methoxyethyl)-3′-phosphonoacetate (MOEP), or a 2′-O-(2-methoxyethyl)-3′-thiophosphonoacetate (MOESP).
A synthetic crRNA comprising a guide sequence capable of hybridizing to a target polynucleotide that comprises a target sequence adjacent to a PAM site, wherein said guide sequence consists of 20-N nucleotides, wherein N is an integer between −10 and 3;
- and wherein the guide sequence comprises at least one modification located at position 4-N, 5-N, 7-N, 9-N, 10-N or 11-N relative to the 5′-end of the guide sequence, or a combination thereof;
- wherein the synthetic crRNA has enhanced specificity for the target sequence compared to a crRNA without said modification, and wherein said at least one modification is a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′-thiophosphonoacetate (MSP), a 2′-O-methyl-3′-phosphorothioate (MS), a 2′-O-(2-methoxyethyl) (MOE), a 2′-O-(2-methoxyethyl)-3′-phosphorothioate (MOES), a 2′-O-(2-methoxyethyl)-3′-phosphonoacetate (MOEP), or a 2′-O-(2-methoxyethyl)-3′-thiophosphonoacetate (MOESP).
Dependencies are inferred from the claim text.