7S4U

Cryo-EM structure of Cas9 in complex with 12-14MM DNA substrate, 5 minute time-point

7S4U の概要

• エントリーDOI: 10.2210/pdb7s4u/pdb
• EMDBエントリー: 24833 24834 24835 24836 24837 24838
• 分子名称: CRISPR-associated endonuclease Cas9/Csn1, gRNA, Target strand, ... (4 entities in total)
• 機能のキーワード: cas9, mismatch, 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: 206303.02

構造登録者

Bravo, J.P.K.,Taylor, D.W.,Liu, M.S.,Johnson, K.A. (登録日: 2021-09-09, 公開日: 2022-03-02, 最終更新日: 2024-06-05)

主引用文献

Bravo, J.P.K.,Liu, M.S.,Hibshman, G.N.,Dangerfield, T.L.,Jung, K.,McCool, R.S.,Johnson, K.A.,Taylor, D.W.
Structural basis for mismatch surveillance by CRISPR-Cas9.
Nature, 603:343-347, 2022

PubMed Abstract: CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 variants with greater discrimination against mismatches have been designed, these suffer from substantially reduced rates of on-target DNA cleavage. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

PubMed: 35236982
DOI: 10.1038/s41586-022-04470-1

実験手法

ELECTRON MICROSCOPY (3.56 Å)