8UZB

Cryo-EM structure of iGeoCas9 in complex with sgRNA and target DNA

8UZB の概要

• エントリーDOI: 10.2210/pdb8uzb/pdb
• 関連するPDBエントリー: 8UZA
• EMDBエントリー: 42837 42838
• 分子名称: CRISPR-associated endonuclease Cas9, RNA (107-MER), Target strand DNA (39-MER), ... (4 entities in total)
• 機能のキーワード: rna-guided dna endonuclease, ternary complex, crispr, hydrolase, hydrolase-rna-dna complex, hydrolase/rna/dna
• 由来する生物種: Geobacillus stearothermophilus; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 202904.34

構造登録者

Eggers, A.R.,Soczek, K.M.,Tuck, O.T.,Doudna, J.A. (登録日: 2023-11-14, 公開日: 2024-05-29, 最終更新日: 2024-07-03)

主引用文献

Eggers, A.R.,Chen, K.,Soczek, K.M.,Tuck, O.T.,Doherty, E.E.,Xu, B.,Trinidad, M.I.,Thornton, B.W.,Yoon, P.H.,Doudna, J.A.
Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.
Cell, 187:3249-, 2024

PubMed Abstract: Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

PubMed: 38781968
DOI: 10.1016/j.cell.2024.04.031

実験手法

ELECTRON MICROSCOPY (2.63 Å)