CRISPR GUARD: short guide RNAs protect off-target sites from Cas9 nuclease activity

Precise genome editing using CRISPR-Cas9 driven nucleases and base editors are promising therapeutic avenues for genetic diseases, although collateral damage caused by off-targets is a significant safety concern1–10. Short guide RNAs less than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity11–13. Here we describe CRISPR GUide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting known or predicted off-targets sites by co-delivery of short guide RNAs directed against the off-target loci to compete with the on-target guide RNA. Using this approach, we reduce off-target mutagenesis at multiple off-target sites simultaneously, while retaining on-target editing efficiency. We explore rules for optimal protection of off-target sites with CRISPR GUARD including guide RNA design and dosing. We reveal CRISPR GUARD as a rapidly implementable and general strategy to improve the specificity of genome editing with CRISPR-Cas9 and base editors.