Coupled catalytic states and the role of metal coordination in Cas9
Controlling the activity of the CRISPR–Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remai...
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| Published in: | Nature catalysis Vol. 6; no. 10; pp. 969 - 977 |
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| Main Authors: | , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
London
Nature Publishing Group UK
01-10-2023
Springer Nature |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Controlling the activity of the CRISPR–Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remain elusive. Here we report five cryo-electron microscopy structures of the active
Acidothermus cellulolyticus
Cas9 complex along the reaction path at 2.2–2.9 Å resolution. We observed that a large movement in one nuclease domain, triggered by the cognate DNA, results in noticeable changes in the active site of the other domain that is required for metal coordination and catalysis. Furthermore, the conformations synchronize the reaction intermediates, enabling coupled cutting of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments to the metal-coordination residues lead to altered metal specificity of
A. cellulolyticus
Cas9 and commonly used
Streptococcus pyogenes
Cas9 in cells.
Cas9 is a powerful genome-manipulation enzyme, although how its catalytic activity is controlled is not completely solved. Now, cryo-electron microscopy structures of
Acidothermus cellulolyticus
Cas9 provide atomic-level insights into its activation involving DNA binding, conformational changes and the formation of its two active sites. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 USDOE Office of Science (SC) SC0012704 A.D. and H.L. designed the experiments. A.D. purified the samples. J.R. and A.D. prepared the cryo-EM grids and the collected data. J.R. performed the cryo-EM analysis. M.O.R., M.L.M. and M.R.B. made the mutants and performed the cell survival assays. A.D. performed the in vitro assays with the assistance of Y.S. The data were analysed by A.D., J.R. and H.L., who also made the figures and wrote the manuscript. All authors edited the manuscript. Author contributions |
| ISSN: | 2520-1158 2520-1158 |
| DOI: | 10.1038/s41929-023-01031-1 |