Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

Conference · Thu May 01 04:00:00 EDT 2025

DOI:https://doi.org/10.1016/j.molcel.2025.03.024· OSTI ID:2573989

Shi, Honglue; Al-Sayyad, Noor; Wasko, Kevin M; Trinidad, Marena I; Doherty, Erin E; Vohra, Kamakshi; Boger, Ron S; Colognori, David; Cofsky, Joshua C; Skopintsev, Petr; Bryant, Zev; Doudna, Jennifer A

RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next 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 Streptococcus 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.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: US Department of Energy; USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
• DOE Contract Number: AC02-05CH11231
• OSTI ID: 2573989
• Resource Type: Conference paper
• Conference Information: Molecular Cell
• Country of Publication: United States
• Language: English