- EMDB-24834: Cas9 in complex with 12-14MM DNA at 60 minute time-point, linear ... -
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Open data
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Basic information
Entry
Database: EMDB / ID: EMD-24834
Title
Cas9 in complex with 12-14MM DNA at 60 minute time-point, linear conformation
Map data
Cas9 in complex with 12-14MM DNA at 60 minute time-point, linear conformation
Sample
Complex: SARS-CoV-2 RdRp complex with template:primer and four RMP
Function / homology
Function and homology information
maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding Similarity search - Function
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM138348
United States
Citation
Journal: Nature / Year: 2022 Title: Structural basis for mismatch surveillance by CRISPR-Cas9. Authors: Jack P K Bravo / Mu-Sen Liu / Grace N Hibshman / Tyler L Dangerfield / Kyungseok Jung / Ryan S McCool / Kenneth A Johnson / David W Taylor / 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 ...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.
History
Deposition
Sep 9, 2021
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Header (metadata) release
Mar 2, 2022
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Map release
Mar 2, 2022
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Update
Mar 23, 2022
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Current status
Mar 23, 2022
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
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