Chimeric CRISPR guides enhance Cas9 target specificity

Oligonucleotide-guided nucleases (OGNs) have enabled rapid advances in precision genome engineering. Though much effort has gone into characterizing and mitigating mismatch tolerance for the most widely adopted OGN, Streptococcus pyogenes Cas9 (SpCas9), potential off-target interactions may still limit applications where on-target specificity is critical. Here we present a new axis to control mismatch sensitivity along the recognition-conferring spacer sequence of SpCas9s guide RNA (gRNA). We introduce mismatch-evading loweredthermostability guides (melt-guides) and exhibit how nucleotide-type substitutions in the spacer can reduce cleavage of sequences mismatched by as few as a single base pair. Cotransfecting melt-guides into human cell culture with an exonuclease involved in DNA repair, we observe indel formation on a standard genomic target at approximately 70% the rate of canonical gRNA and undetectable on off-target data.