Structures of Cas9 endonucleases reveal RNA-mediated conformational activation
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Introduction Bacteria and archaea defend themselves against invasive DNA using adaptive immune systems comprising CRISPR (clustered regularly interspaced short palindromic repeats) loci and CRISPR-associated (Cas) genes. In association with Cas proteins, small CRISPR RNAs (crRNAs) guide the detection and cleavage of complementary DNA sequences. Type II CRISPR systems employ the RNA-guided endonuclease Cas9 to recognize and cleave double-stranded DNA (dsDNA) targets using conserved RuvC and HNH nuclease domains. Cas9-mediated cleavage is strictly dependent on the presence of a protospacer adjacent motif (PAM) in the target DNA. Recently, the biochemical properties of Cas9–guide RNA complexes have been harnessed for various genetic engineering applications and RNA-guided transcriptional control. Despite these ongoing successes, the structural basis for guide RNA recognition and DNA targeting by Cas9 is still unknown. Rationale To compare the architectures and domain organization of diverse Cas9 proteins, the atomic structures of Cas9 from Streptococcus pyogenes (SpyCas) and Actinomyces naeslundii (AnaCas9) were determined by x-ray crystallography. Crosslinking of target DNA containing 5-bromodeoxyuridines was conducted to identify PAM-interacting regions in SpyCas9. To test functional interactions with nucleic acid ligands, structure-based mutant SpyCas9 proteins were assayed for endonuclease activity with radiolabeled oligonucleotide dsDNA targets, and target DNA binding was monitored by electrophoretic mobility shift assays. To compare conformations of Cas9 in different states of nucleic acid binding, three-dimensional reconstructions of apo-SpyCas9, SpyCas9:RNA, and SpyCas9:RNA:DNA were obtained by negative-stain single-particle electron microscopy. Guide RNA and target DNA positions were determined with streptavidin labeling. Exonuclease protection assays were carried out to determine the extent of Cas9–target DNA interactions. Results The 2.6 Å–resolution structure of apo-SpyCas9 reveals a bilobed architecture comprising a nuclease domain lobe and an α-helical lobe. Both lobes contain conserved clefts that may function in nucleic acid binding. Photocrosslinking experiments show that the PAM in target DNA is engaged by two tryptophan-containing flexible loops, and mutations of both loops impair target DNA binding and cleavage. The 2.2 Å–resolution crystal structure of AnaCas9 reveals the conserved structural core shared by all Cas9 enzyme subtypes, and both SpyCas9 and AnaCas9 adopt autoinhibited conformations in their apo forms. The electron microscopic (EM) reconstructions of SpyCas9:RNA and SpyCas9:RNA:DNA complexes reveal that guide RNA binding results in a conformational rearrangement and formation of a central channel for target DNA binding. Site-specific labeling of guide RNA and target DNA define the orientations of nucleic acids in the target-bound complex. Conclusion The SpyCas9 and AnaCas9 structures define the molecular architecture of the Cas9 enzyme family in which a conserved structural core encompasses the two nuclease domains responsible for DNA cleavage, while structurally divergent regions, including the PAM recognition loops, are likely responsible for distinct guide RNA and PAM specificities. Cas9 enzymes adopt a catalytically inactive conformation in the apo state, necessitating structural activation for DNA recognition and cleavage. Our EM analysis shows that by triggering a conformational rearrangement in Cas9, the guide RNA acts as a critical determinant of target DNA binding.
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Native Cas9 protein is positively charged, while conventional proteins are negatively charged.
“Meanwhile, native Cas9 protein is positively charged, while the conventional proteins are negatively charged [88, 97, 103, 139].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [139]
First obstacle: effective encapsulation of editing tools in delivery vectors is hindered by large size and different charge properties of Cas9 protein, mRNA and donor DNA.
“The first obstacle is effective encapsulation of the editing tools in delivery vectors due to the large size and different charge properties of Cas9 protein, mRNA and donor DNA.”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [139]
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DNA Binding and Cleavage by the HNH Homing Endonuclease I-HmuI
Shen, B. W., Landthaler, M., Shub, D. A., & Stoddard, B. L. (2004) · Journal of Molecular Biology · doi:10.1016/j.jmb.2004.07.032 · Cited by 130 · see citing works
Analysis of CRISPR system function in plant pathogenXanthomonas oryzae
Semenova, E., Nagornykh, M. O., Pyatnitskiy, M. A., Artamonova, I. I., & Severinov, K. (2009) · FEMS Microbiology Letters · doi:10.1111/j.1574-6968.2009.01626.x · Cited by 75 · see citing works
Crystal structure of RuvC resolvase in complex with Holliday junction substrate
Górecka, K. M., Komorowska, W., & Nowotny, M. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt769 · Cited by 70 · see citing works
Nartey, C. M., Nam, K., Oo, K., Kutchukian, P. S., Bowman, B. R., Karplus, M., & Verdine, G. L. (2012) · Journal of Biological Chemistry · doi:10.1074/jbc.m111.316497 · Cited by 54 · see citing works
A Toolbox for ab initio 3-D reconstructions in single-particle electron microscopy
Voss, N. R., Lyumkis, D., Cheng, A., et al. (2009) · Journal of Structural Biology · doi:10.1016/j.jsb.2009.12.005 · Cited by 46 · see citing works
Tuning in to Interference: R-Loops and Cascade Complexes in CRISPR Immunity
Ivančić-Baće, I., Howard, J. A. L., & Bolt, E. L. (2012) · Journal of Molecular Biology · doi:10.1016/j.jmb.2012.06.024 · Cited by 29 · see citing works
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The new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062 · see citing works
Self-citationDevelopment and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762 · see citing works
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 · see citing works
CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity
Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018) · Science · doi:10.1126/science.aar6245 · Cited by 4,507 · see citing works
Self-citationHigh-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 · 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
A CRISPR/Cas9 toolkit for multiplex genome editing in plants
Xing, H.-L., Dong, L., Wang, Z.-P., Zhang, H.-Y., Han, C.-Y., Liu, B., Wang, X.-C., & Chen, Q.-J. (2014) · BMC Plant Biology · doi:10.1186/s12870-014-0327-y · Cited by 1,684 · see citing works
Diversity, classification and evolution of CRISPR-Cas systems
Koonin, E. V., Makarova, K. S., & Zhang, F. (2017) · Current Opinion in Microbiology · doi:10.1016/j.mib.2017.05.008 · Cited by 1,423 · see citing works
Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S., & Yang, S.-H. (2015) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2015.37 · Cited by 1,273 · see citing works
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
Chen, J. S., Dagdas, Y. S., Kleinstiver, B. P., Welch, M. M., Sousa, A. A., Harrington, L. B., Sternberg, S. H., Joung, J. K., Yildiz, A., & Doudna, J. A. (2017) · Nature · doi:10.1038/nature24268 · Cited by 1,168 · see citing works
Self-citationHigh-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
Delivering CRISPR: a review of the challenges and approaches
Lino, C. A., Harper, J. C., Carney, J. P., & Timlin, J. A. (2018) · Drug Delivery · doi:10.1080/10717544.2018.1474964 · Cited by 1,185 · 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
Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
Tsai, S. Q., Wyvekens, N., Khayter, C., Foden, J. A., Thapar, V., Reyon, D., Goodwin, M. J., Aryee, M. J., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2908 · Cited by 952 · see citing works
Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds
Zalatan, J. G., Lee, M. E., Almeida, R., Gilbert, L. A., Whitehead, E. H., La Russa, M., Tsai, J. C., Weissman, J. S., Dueber, J. E., Qi, L. S., et al. (2015) · Cell · doi:10.1016/j.cell.2014.11.052 · Cited by 938 · see citing works
Guilinger, J. P., Thompson, D. B., & Liu, D. R. (2014) · Nature Biotechnology · doi:10.1038/nbt.2909 · Cited by 836 · see citing works
Conformational control of DNA target cleavage by CRISPR–Cas9
Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A. (2015) · Nature · doi:10.1038/nature15544 · Cited by 627 · see citing works
Self-citationA Multifunctional AAV-CRISPR-Cas9 and Its Host Response
Chew, W. L., Tabebordbar, M., Cheng, J. K. W., Mali, P., Wu, E. Y., Ng, A. H. M., Zhu, K., Wagers, A. J., & Church, G. M. (2016) · Nature Methods · doi:10.1038/nmeth.3993 · Cited by 617 · see citing works
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition
Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Topkar, V. V., Zheng, Z., & Joung, J. K. (2015) · Nature Biotechnology · doi:10.1038/nbt.3404 · Cited by 611 · see citing works
CRISPR/Cas system: recent advances and future prospects for genome editing
Manghwar, H., Lindsey, K., Zhang, X., & Jin, S. (2019) · Trends in Plant Science · doi:10.1016/j.tplants.2019.09.006 · Cited by 529 · see citing works
“pyogenes and comprises three components: the CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and a Cas9 protein [99].”
BackgroundOrigins 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
Classification and evolution of type II CRISPR-Cas systems
Chylinski, K., Makarova, K. S., Charpentier, E., & Koonin, E. V. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku241 · Cited by 498 · see citing works
Self-citationDNA 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
The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases
Altae-Tran, H., Kannan, S., Demircioglu, F. E., Oshiro, R., Nety, S. P., McKay, L. J., Dlakić, M., Inskeep, W. P., Makarova, K. S., Macrae, R. K., et al. (2021) · Science · doi:10.1126/science.abj6856 · Cited by 407 · see citing works
Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities
Li, L., Hu, S., & Chen, X. (2018) · Biomaterials · doi:10.1016/j.biomaterials.2018.04.031 · Cited by 415 · see citing works
Developmental barcoding of whole mouse via homing CRISPR
Kalhor, R., Kalhor, K., Mejia, L., Leeper, K., Graveline, A., Mali, P., & Church, G. M. (2018) · Science · doi:10.1126/science.aat9804 · Cited by 363 · see citing works
Engineering the delivery system for CRISPR-based genome editing
Glass, Z., Lee, M., Li, Y., & Xu, Q. (2018) · Trends in biotechnology · doi:10.1016/j.tibtech.2017.11.006 · Cited by 345 · see citing works
Zischewski, J., Fischer, R., & Bortesi, L. (2017) · Biotechnology Advances · doi:10.1016/j.biotechadv.2016.12.003 · Cited by 323 · see citing works
Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d
Zhang, C., Konermann, S., Brideau, N. J., Lotfy, P., Wu, X., Novick, S. J., Strutzenberg, T., Griffin, P. R., Hsu, P. D., & Lyumkis, D. (2018) · Cell · doi:10.1016/j.cell.2018.09.001 · Cited by 276 · see citing works
A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9
Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A., & Yildiz, A. (2017) · Science Advances · doi:10.1126/sciadv.aao0027 · Cited by 293 · see citing works
Self-citationStructural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9
Anders, C., Bargsten, K., & Jinek, M. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.02.020 · Cited by 201 · see citing works
Self-citationStructural basis for the altered PAM specificities of engineered CRISPR-cas9
Hirano, S., Nishimasu, H., Ishitani, R., & Nureki, O. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.02.018 · Cited by 152 · see citing works
Genome-wide identification of CRISPR/Cas9 off-targets in human genome
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RNA-Targeting CRISPR-Cas systems and their applications
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Self-citationCharacterization and Repurposing of Type I and Type II CRISPR–Cas Systems in Bacteria
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Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models
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- Authors
- Jinek, M., Jiang, F., Taylor, D. W., Sternberg, S. H., Kaya, E., Ma, E., Anders, C., Hauer, M., Zhou, K., Lin, S., Kaplan, M., Iavarone, A. T., Charpentier, E., Nogales, E., & Doudna, J. A.
- Affiliations
- Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.
- University of California, Berkeley (US)
- QB3 (US)
- University of Zurich (CH)
- Howard Hughes Medical Institute (US)
- Medizinische Hochschule Hannover (DE)
- Corresponding author
- Jiang, Fuguo
- Taylor, David W.
- Sternberg, Samuel H.
- Hauer, Michael
- Kaplan, Matias
- Nogales, Eva
- Doudna, Jennifer A.
- ORCID
- Venue
- Science
- Published
- 2014-03-14
- Volume
- 343
- Issue
- 6176
- Pages
- 1247997-1247997
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC4184034
- OpenAlex
- W2142515819
- Cited by
- 1,230
- References
- 87
- Citations by year
- 2026: 55 · 2025: 94 · 2024: 110 · 2023: 100 · 2022: 115 · 2021: 102 · 2020: 109 · 2019: 103 · 2018: 95 · 2017: 98 · 2016: 95 · 2015: 78 · 2014: 72 · 2013: 2
- FWCI
- 46.91
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Science (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DORA PSI (Paul Scherrer Institute) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of General Medical Sciences (T32GM066698)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- Cas9, CRISPR/Cas9, RNA-guided DNA cleavage, conformational activation, protospacer adjacent motif, PAM recognition, SpCas9, nuclease domain, HNH nuclease, single-particle electron microscopy
- MeSH terms
- Actinomyces, Amino Acid Sequence, Bacterial Proteins, Endonucleases, Molecular Sequence Data, Nucleic Acid Conformation, RNA, Streptococcus pyogenes, Protein Structure, Secondary, Protein Structure, Tertiary, Crystallography, X-Ray, DNA Cleavage, Clustered Regularly Interspaced Short Palindromic Repeats
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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.
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