9K4E

SuperFi Cas9 - mismatch 22nt sgRNA - DNA ternary complex class1

9K4E の概要

• エントリーDOI: 10.2210/pdb9k4e/pdb
• 関連するPDBエントリー: 9K4C 9K4D
• EMDBエントリー: 62054 62055 62056
• 分子名称: CRISPR-associated endonuclease Cas9/Csn1, DNA (50-MER), RNA (102-MER), ... (4 entities in total)
• 機能のキーワード: complex, cas, superfi cas9, immune system, immune system-dna-rna complex, immune system/dna/rna
• 由来する生物種: Streptococcus pyogenes; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 222088.50

構造登録者

Zheng, R.,Ma, L.J. (登録日: 2024-10-21, 公開日: 2025-10-22, 最終更新日: 2026-05-20)

主引用文献

Zheng, R.,Lu, Z.,Wei, R.,Shin, Y.C.,Du, J.,Zhang, Q.,Li, J.,Wang, X.,Wei, Y.,Liu, B.,Chen, Y.,Ding, L.,Zhang, H.,Chen, H.,Huang, J.,Ma, L.
Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions.
Nat.Struct.Mol.Biol., 33:590-602, 2026

PubMed Abstract: Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.

PubMed: 41851507
DOI: 10.1038/s41594-026-01753-3

実験手法

ELECTRON MICROSCOPY (3.3 Å)