Shi, H.; Al-Sayyad, N.; Wasko, K.; Trinidad, M. I.; Doherty, E. E.; Vohra, K.; Boger, R. S.; Colognori, D.; Cofsky, J. C.; Skopintsev, P.; Bryant, Z.; Doudna, J. A.

RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on S. pyogenes Cas9 (SpyCas9) variants with wide-ranging genome editing efficiencies that differ in PAM binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.