Structure of Geobacillus stearothermophilus Cas9: Insights into the Catalytic Process and Thermostability of CRISPR-Cas9

被引：3

作者：

Shen, Panpan ^{[1
]}

Zhang, Lilan ^{[1
]}

Liu, Beibei ^{[1
]}

Li, Xian ^{[1
]}

Wang, Caihong ^{[1
]}

Min, Jian ^{[1
]}

Huang, Jian-Wen ^{[1
,2
]}

Chen, Chun-Chi ^{[1
,2
]}

Guo, Rey-Ting ^{[1
,2
]}

机构：

[1] Hubei Univ, Hubei Collaborat Innovat Ctr Green Transformat Bio, Sch Life Sci, Hubei Key Lab Ind Biotechnol,State Key Lab Biocata, Wuhan 430062, Peoples R China

[2] Hangzhou Normal Univ, Sch Basic Med Sci, Dept Immunol & Pathogen Biol, Zhejiang Key Lab Med Epigenet, Hangzhou 311121, Peoples R China

来源：

ACS CATALYSIS | 2024年 / 14卷 / 17期

基金：

中国国家自然科学基金;

关键词：

CRISPR-Cas9; cryo-electron microscopy; Geobacillusstearothermophilus; thermostability; catalytic mechanism; L1-crevice; CRYSTAL-STRUCTURE; CRYO-EM; RNA; ENDONUCLEASE; DIVERSITY; COMPLEX; DESIGN;

D O I：

10.1021/acscatal.4c03278

中图分类号：

O64 [物理化学（理论化学）、化学物理学];

学科分类号：

070304 ; 081704 ;

摘要：

CRISPR-Cas9 has been developed as a powerful gene editing tool, but the mechanism that governs its intricate catalytic process remains incompletely resolved. Here, the cryo-electron microscopy structures of thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) in a complex with sgRNA and target DNA are reported. The structure of GeoCas9 in a complex with sgRNA reveals a slit termed an L1 crevice comprising HNH, RuvC, and an L1 helix as a transient storage site of the 5 ' spacer fragment of sgRNA. When the loosely bound 5 ' spacer segment is extracted to pair with the target DNA, the L1 crevice collapses to trigger subsequent HNH domain translocation. In addition, structural and biochemical analyses suggest that the resilience of GeoCas9 at elevated temperatures is related to the unique PI domain conformation and a higher proline content of the WED domain. These results advance our understanding of the catalytic process of Cas9 and unveil the molecular mechanism that accounts for the superior thermal profile of GeoCas9.

引用

页码：13227 / 13235

页数：9

共 48 条

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