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

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.

doi:10.1101/2024.10.01.616117

bioRxiv · 10.1101/2024.10.01.616117

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

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.

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Abstract

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.

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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.. 2024-10-02. Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing. https://doi.org/10.1101/2024.10.01.616117

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