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US 12,655,422 B2Granted
Methods and compositions for inhibition of CRISPR re-cleavage events
AssigneeIntegrated Dna Technologies, Inc.
InventorsGarrett R. Rettig, Nathan Delvaux, Ashley M. Jacobi, Mollie S Schubert, Nathanial Roberts, Mark A Behlke
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Abstract
The disclosure relates to methods and compositions that serve to reduce the potential for targeted nuclease activity for example, but not limited to, CRISPR enzymes such as Cas9 and/or Cas12a. The disclosure also relates to methods and compositions be used as an HDR substrate to prevent Cas9 re-cleavage, and as a result increase the frequency of perfect HDR.
Claims13
A modified double stranded DNA homology directed repair (HDR) donor comprising:
- a targeting strand comprising one or more internal chemical modifications selected from the group consisting of:
- Locked Nucleic Acid (LNA), 2′-O-methoxyethyl (2′-MOE), and 2′-O-methyl (2′-OMe), and further wherein the one or more internal chemical modifications are adjacent to the endonuclease cleavage site;
- a non-targeting strand, wherein the non-targeting strand lacks the one or more internal chemical modifications comprised by the targeting strand,
- wherein the one or more internal chemical modifications on the targeting strand reduces a nuclease-mediated re-cleavage and improves HDR efficiency.
1 more element
Dependencies are inferred from the claim text.