Structural basis for mismatch surveillance by CRISPR–Cas9

Bravo JPK, Liu M-S, Hibshman GN, Dangerfield TL, Jung K, McCool RS, Johnson KA, Taylor DW. 2022. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature. 603(7900), 343–347.

Download (ext.)

Journal Article | Published | English

Scopus indexed
Author
Bravo, JackISTA ; Liu, Mu-Sen; Hibshman, Grace N.; Dangerfield, Tyler L.; Jung, Kyungseok; McCool, Ryan S.; Johnson, Kenneth A.; Taylor, David W.
Abstract
CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1,2,3,4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5,6,7. Although Cas9 variants with greater discrimination against mismatches have been designed8,9,10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. 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.
Publishing Year
Date Published
2022-03-02
Journal Title
Nature
Volume
603
Issue
7900
Page
343-347
ISSN
eISSN
IST-REx-ID

Cite this

Bravo JPK, Liu M-S, Hibshman GN, et al. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature. 2022;603(7900):343-347. doi:10.1038/s41586-022-04470-1
Bravo, J. P. K., Liu, M.-S., Hibshman, G. N., Dangerfield, T. L., Jung, K., McCool, R. S., … Taylor, D. W. (2022). Structural basis for mismatch surveillance by CRISPR–Cas9. Nature. Springer Nature. https://doi.org/10.1038/s41586-022-04470-1
Bravo, Jack Peter Kelly, Mu-Sen Liu, Grace N. Hibshman, Tyler L. Dangerfield, Kyungseok Jung, Ryan S. McCool, Kenneth A. Johnson, and David W. Taylor. “Structural Basis for Mismatch Surveillance by CRISPR–Cas9.” Nature. Springer Nature, 2022. https://doi.org/10.1038/s41586-022-04470-1.
J. P. K. Bravo et al., “Structural basis for mismatch surveillance by CRISPR–Cas9,” Nature, vol. 603, no. 7900. Springer Nature, pp. 343–347, 2022.
Bravo JPK, Liu M-S, Hibshman GN, Dangerfield TL, Jung K, McCool RS, Johnson KA, Taylor DW. 2022. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature. 603(7900), 343–347.
Bravo, Jack Peter Kelly, et al. “Structural Basis for Mismatch Surveillance by CRISPR–Cas9.” Nature, vol. 603, no. 7900, Springer Nature, 2022, pp. 343–47, doi:10.1038/s41586-022-04470-1.
All files available under the following license(s):
Copyright Statement:
This Item is protected by copyright and/or related rights. [...]

Link(s) to Main File(s)
Access Level
OA Open Access
External material:
Erratum

Export

Marked Publications

Open Data ISTA Research Explorer

Sources

PMID: 35236982
PubMed | Europe PMC

Search this title in

Google Scholar