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

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

Ngôn ngữ: eng

Ký hiệu phân loại: 109 Historical and collected persons treatment of philosophy

Thông tin xuất bản: United States : Molecular cell , 2025

Mô tả vật lý:

Bộ sưu tập: NCBI

ID: 743799

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.
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