The DOI matches its Crossref record; 15 of the 15 references it cites were found in a registry or an index.
R-loop formation and conformational activation mechanisms of Cas9
Open accessAbstract
Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage 1–3 . The programmable activity of Cas9 has been widely utilized for genome editing applications 4–6 , yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide–target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide–target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide–target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.
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Citations
Cites
RNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016) · Nature · doi:10.1038/nature17946 · Cited by 5,437
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016
DNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3437 · Cited by 4,637
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017) · Nature · doi:10.1038/nature24644 · Cited by 4,184
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., & Joung, J. K. (2016) · Nature · doi:10.1038/nature16526 · Cited by 2,673
Crystal structure of Cas9 in complex with guide RNA and target DNA
Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014) · Cell · doi:10.1016/j.cell.2014.02.001 · Cited by 2,306
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3117 · Cited by 2,268
Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
Anzalone, A. V., Koblan, L. W., & Liu, D. R. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0561-9 · Cited by 2,237
Cited by
CRISPR/Cas9 therapeutics: progress and prospects
Li, T., Yang, Y., Qi, H., Cui, W., Zhang, L., Fu, X., He, X., Liu, M., Li, P.-F., & Yu, T. (2023) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-023-01309-7 · Cited by 719
Past, present, and future of CRISPR genome editing technologies
Pacesa, M., Pelea, O., & Jinek, M. (2024) · Cell · doi:10.1016/j.cell.2024.01.042 · Cited by 609
Structural basis for Cas9 off-target activity
Pacesa, M., Lin, C.-H., Cléry, A., Saha, A., Arantes, P. R., Bargsten, K., Irby, M. J., Allain, F. H.-T., Palermo, G., Cameron, P., et al. (2022) · Cell · doi:10.1016/j.cell.2022.09.026 · Cited by 198
Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences
Ruffolo, J. A., Nayfach, S., Gallagher, J., Bhatnagar, A., Beazer, J., Hussain, R., Russ, J., Yip, J., Hill, E., Pacesa, M., et al. (2024) · doi:10.1101/2024.04.22.590591 · Cited by 90
G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation
Deng, H., Xu, H., Wang, Y., Jia, R., Ma, X., Feng, Y., & Chen, H. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad178 · Cited by 45
Metadata
- Authors
- Pacesa, M., Loeff, L., Querques, I., Muckenfuss, L. M., Sawicka, M., & Jinek, M.
- Venue
- Nature
- Published
- 2022-08-24
- Volume
- 609
- Issue
- 7925
- Pages
- 191-196
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- OpenAlex
- W4293004809
- Cited by
- 169
- References
- 15
- FWCI
- 9.25
- Type
- journal article
- Language
- en
- Open access
- hybrid
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