CRISPR RNA architecture steers Cas9 catalysis and fidelity via a guide repeat clasp motif

A widely adopted modification of CRISPR-Cas9 is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). Here we find that these guide architectures induce differential catalysis, gene editing, and specificity. Spacer sequence and RNA structural features could not predict guide architecture editing preference across 255 endogenous targets. We used cryo-EM and molecular dynamics to identify a new Cas9 structural motif, the guide repeat clasp (GRC), that checks guide RNA repeat structure and coordinates with R-loop sensing checkpoint mechanisms to help license cleavage. Limited mutagenesis of GRC residues significantly altered Cas9 editing and specificity, supporting a key role in catalysis. To further understand the role of the GRC and guide RNA repeat dynamics, we created guide repeat-truncated sgRNAs, or grtRNAs, which conferred some dgRNA properties onto sgRNA, including generally lower off-target editing for targets with PAM-proximal mismatches. dgRNAs and grtRNAs could be combined with a new high-fidelity Cas9 variant called ZiFY, rationally designed to reduce editing of targets with PAM-distal mismatches, to generate broader mismatched target discrimination. These results uncover a previously unknown mechanism that steers Cas9 catalysis and demonstrate the potential to improve Cas9 fidelity by modulating guide repeat interactions.