CRISPR–Cas9 Structures and Mechanisms
Abstract
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
SpCas9 requires the 'NGG' PAM sequence, which is commonly found in genomes, enabling CRISPR/Cas9 use in a wide range of species.
“SpCas9 requires “NGG” as a PAM sequence, which is commonly found in the genomes of most organisms, enabling the use of CRISPR/Cas9 technology in a wide range of species (Jiang & Doudna, 2017).”
From CRISPR/Cas9 Ribonucleoprotein Nucleofection for Genome Editing in Primary Human Keratinocytes: Knockouts, Deletions, and Homology‐Directed Repair Mutagenesis (2024) · cites this paper as Jiang & Doudna, 2017
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Self-citationMultiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
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 · see citing works
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292 · see citing works
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153 · see citing works
The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway
Lieber, M. R. (2010) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.052308.093131 · Cited by 2,882 · see citing works
In vivo genome editing using Staphylococcus aureus Cas9
Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., et al. (2015) · Nature · doi:10.1038/nature14299 · Cited by 2,687 · see citing works
RNA-guided editing of bacterial genomes using CRISPR-Cas systems
Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013) · Nature Biotechnology · doi:10.1038/nbt.2508 · Cited by 2,429 · see citing works
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 · see citing works
RNA-programmed genome editing in human cells
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013) · eLife · doi:10.7554/elife.00471 · Cited by 2,098 · see citing works
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2808 · Cited by 1,993 · see citing works
RNA-guided genetic silencing systems in bacteria and archaea
Wiedenheft, B., Sternberg, S. H., & Doudna, J. A. (2012) · Nature · doi:10.1038/nature10886 · Cited by 1,851 · see citing works
Self-citationEngineered CRISPR-Cas9 nucleases with altered PAM specificities
Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., Gonzales, A. P. W., Li, Z., Peterson, R. T., Yeh, J.-R. J., et al. (2015) · Nature · doi:10.1038/nature14592 · Cited by 1,624 · see citing works
Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R. (2013) · Nature Biotechnology · doi:10.1038/nbt.2673 · Cited by 1,487 · see citing works
High-throughput functional genomics using CRISPR-Cas9
Shalem, O., Sanjana, N. E., & Zhang, F. (2015) · Nature Reviews Genetics · doi:10.1038/nrg3899 · Cited by 1,233 · see citing works
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
Wu, X., Scott, D. A., Kriz, A. J., Chiu, A. C., Hsu, P. D., Dadon, D. B., Cheng, A. W., Trevino, A. E., Konermann, S., Chen, S., et al. (2014) · Nature Biotechnology · doi:10.1038/nbt.2889 · Cited by 944 · see citing works
Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation
Dominguez, A. A., Lim, W. A., & Qi, L. S. (2016) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm.2015.2 · Cited by 832 · see citing works
Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage
Cencic, R., Miura, H., Malina, A., Robert, F., Ethier, S., Schmeing, T. M., Dostie, J., & Pelletier, J. (2014) · PLoS ONE · doi:10.1371/journal.pone.0109213 · Cited by 117 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Origins and evolution of CRISPR-Cas systems
Koonin, E. V., & Makarova, K. S. (2019) · Philosophical Transactions of the Royal Society B: Biological Sciences · doi:10.1098/rstb.2018.0087 · Cited by 494 · see citing works
DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing
Xue, C., & Greene, E. C. (2021) · Trends in Genetics · doi:10.1016/j.tig.2021.02.008 · Cited by 431 · see citing works
Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs
Ryan, D. E., Taussig, D., Steinfeld, I., Phadnis, S. M., Lunstad, B. D., Singh, M., Vuong, X., Okochi, K. D., McCaffrey, R., Olesiak, M., et al. (2018) · Nucleic Acids Research · doi:10.1093/nar/gkx1199 · Cited by 280 · see citing works
CRISPR-Cas9 for cancer therapy: Opportunities and challenges
Chen, M., Mao, A., Xu, M., Weng, Q., Mao, J., & Ji, J. (2019) · Cancer Letters · doi:10.1016/j.canlet.2019.01.017 · Cited by 272 · see citing works
Chehelgerdi, M., Chehelgerdi, M., Khorramian-Ghahfarokhi, M., Shafieizadeh, M., Mahmoudi, E., Eskandari, F., Rashidi, M., Arshi, A., & Mokhtari-Farsani, A. (2024) · Molecular Cancer · doi:10.1186/s12943-023-01925-5 · Cited by 251 · see citing works
Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes
Hess, G. T., Tycko, J., Yao, D., & Bassik, M. C. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.09.029 · Cited by 234 · see citing works
Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity
Cromwell, C. R., Sung, K., Park, J., Krysler, A. R., Jovel, J., Kim, S. K., & Hubbard, B. P. (2018) · Nature Communications · doi:10.1038/s41467-018-03927-0 · Cited by 190 · see citing works
Current status and perspectives of genome editing technology for microalgae
Jeon, S., Lim, J.-M., Lee, H.-G., Shin, S.-E., Kang, N. K., Park, Y.-I., Oh, H.-M., Jeong, W.-J., Jeong, B.-R., & Chang, Y. K. (2017) · Biotechnology for Biofuels · doi:10.1186/s13068-017-0957-z · Cited by 154 · see citing works
Palermo, G., Chen, J. S., Ricci, C. G., Rivalta, I., Jinek, M., Batista, V. S., Doudna, J. A., & McCammon, J. A. (2018) · Quarterly Reviews of Biophysics · doi:10.1017/s0033583518000070 · Cited by 123 · see citing works
Self-citationCurrent Bioinformatics Tools to Optimize CRISPR/Cas9 Experiments to Reduce Off-Target Effects
Naeem, M., & Alkhnbashi, O. S. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms24076261 · Cited by 70 · see citing works
Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models
Chenouard, V., Remy, S., Tesson, L., Ménoret, S., Ouisse, L.-H., Cherifi, Y., & Anegon, I. (2021) · Frontiers in Genetics · doi:10.3389/fgene.2021.615491 · Cited by 57 · see citing works
Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches
Leal, A., Herreno-Pachón, A., Benincore-Flórez, E., Karunathilaka, A., & Tomatsu, S. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25052456 · Cited by 58 · see citing works
Tariq, A., Mushtaq, M., Yaqoob, H., Bhat, B. A., Zargar, S. M., Raza, A., Ali, S., Charagh, S., Mubarik, M. S., Zaman, Q. U., et al. (2023) · GM Crops & Food · doi:10.1080/21645698.2023.2219111 · Cited by 45 · see citing works
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 · see citing works
Emerging applications of gene editing technologies for the development of climate-resilient crops
Chavhan, R. L., Jaybhaye, S. G., Hinge, V. R., Deshmukh, A. S., Shaikh, U. S., Jadhav, P. K., Kadam, U. S., & Hong, J. C. (2025) · Frontiers in Genome Editing · doi:10.3389/fgeed.2025.1524767 · Cited by 43 · see citing works
Shen, Q., Ruan, H., Zhang, H., Wu, T., Zhu, K., Han, W., Dong, R., Ming, T., Qi, H., & Zhang, Y. (2024) · Frontiers in Microbiology · doi:10.3389/fmicb.2024.1375120 · Cited by 38 · see citing works
Pandey, M., Ojha, D., Bansal, S., Rode, A. B., & Chawla, G. (2021) · Molecular Aspects of Medicine · doi:10.1016/j.mam.2021.101003 · Cited by 23 · see citing works
Translational research to enable personalized treatment of cystic fibrosis
Hagemeijer, M. C., Siegwart, D. J., Strug, L. J., Cebotaru, L., Torres, M. J., Sofoluwe, A., & Beekman, J. M. (2018) · Journal of Cystic Fibrosis · doi:10.1016/j.jcf.2017.10.017 · Cited by 22 · see citing works
Okada, K., Aoki, K., Tabei, T., Sugio, K., Imai, K., Bonkohara, Y., & Kamachi, Y. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac100 · Cited by 14 · see citing works
Bamundo, M., Palumbo, S., D'Auria, L., Missero, C., & Di Girolamo, D. (2024) · Current Protocols · doi:10.1002/cpz1.70056 · Cited by 4 · see citing works
Kinetic mechanism for fidelity of CRISPR-Cas9 variants
Hecht, A. D., & Igoshin, O. A. (2025) · bioRxiv · doi:10.1101/2025.01.27.634389
- Authors
- Jiang, F., & Doudna, J. A.
- Affiliations
- QB3 (US)
- Venue
- Annual Review of Biophysics
- Publisher
- Annual Reviews
- Published
- 2017-05-22
- Volume
- 46
- Issue
- 1
- Pages
- 505-529
- ISSN
- 1936-122X (ISSN-L)
- 1936-1238
- OpenAlex
- W2554763217
- Cited by
- 2,029
- References
- 18
- Citations by year
- 2026: 166 · 2025: 267 · 2024: 296 · 2023: 238 · 2022: 311 · 2021: 270 · 2020: 215 · 2019: 149 · 2018: 82 · 2017: 32
- FWCI
- 12.22
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Annual Review of Biophysics (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Howard Hughes Medical Institute
- Damon Runyon Cancer Research Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- CRISPR/Cas9, protospacer adjacent motif, R-loop formation, guide RNA, single guide RNA, tracrRNA, crRNA, RNA-guided DNA cleavage, phage defense, conjugative plasmids, off-target effects, genome editing
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, DNA, RNA, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
