- EMDB-24836: Cas9 in complex with 15-17MM DNA, 60 min time-point -
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基本情報
登録情報
データベース: EMDB / ID: EMD-24836
タイトル
Cas9 in complex with 15-17MM DNA, 60 min time-point
マップデータ
試料
複合体: Cas9 bound to 15-17MM DNA, 60 min time-point, linear conformation
機能・相同性
機能・相同性情報
maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / 加水分解酵素; エステル加水分解酵素 / DNA binding / RNA binding / metal ion binding 類似検索 - 分子機能
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM138348
米国
引用
ジャーナル: Nature / 年: 2022 タイトル: Structural basis for mismatch surveillance by CRISPR-Cas9. 著者: Jack P K Bravo / Mu-Sen Liu / Grace N Hibshman / Tyler L Dangerfield / Kyungseok Jung / Ryan S McCool / Kenneth A Johnson / David W Taylor / 要旨: 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 ...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.