CRISPR–Cas9 bends and twists DNA to read its sequence

Cofsky, Joshua C.; Soczek, Katarzyna M.; Knott, Gavin J.; Nogales, Eva; Doudna, Jennifer A.

doi:10.1038/s41594-022-00756-0

CRISPR–Cas9 bends and twists DNA to read its sequence

Journal Article · Thu Apr 14 04:00:00 EDT 2022 · Nature Structural & Molecular Biology

DOI:https://doi.org/10.1038/s41594-022-00756-0· OSTI ID:2470935

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Univ. of California, Berkeley, CA (United States)
Monash Univ., Melbourne, VIC (Australia)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Univ. of California, San Francisco, CA (United States); Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA (United States)

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; National Science Foundation (NSF); National Institutes of Health (NIH)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470935

Journal Information:: Nature Structural & Molecular Biology, Journal Name: Nature Structural & Molecular Biology Journal Issue: 4 Vol. 29; ISSN 1545-9993

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (48)

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity Garcia, Hernan G.; Grayson, Paul; Han, Lin Biopolymers, Vol. 85, Issue 2 https://doi.org/10.1002/bip.20627	journal	January 2007
Hhal methyltransferase flips its target base out of the DNA helix Klimasauskas, Saulius; Kumar, Sanjay; Roberts, Richard J. Cell, Vol. 76, Issue 2 https://doi.org/10.1016/0092-8674(94)90342-5	journal	January 1994
The crystal structure of Haelll methyltransferase covalently complexed to DNA: An extrahelical cytosine and rearranged base pairing Reinisch, Karin M.; Chen, Lin; Verdine, Gregory L. Cell, Vol. 82, Issue 1 https://doi.org/10.1016/0092-8674(95)90060-8	journal	July 1995
Atomic model of a pyrimidine dimer excision repair enzyme complexed with a dna substrate: Structural basis for damaged DNA recognition Vassylyev, Dmitry G.; Kashiwagi, Tatsuki; Mikami, Yuriko Cell, Vol. 83, Issue 5 https://doi.org/10.1016/0092-8674(95)90190-6	journal	December 1995
Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex Werner, Milton H.; Huth, Jeffrey R.; Gronenborn, Angela M. Cell, Vol. 81, Issue 5 https://doi.org/10.1016/0092-8674(95)90532-4	journal	June 1995
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA Nishimasu, Hiroshi; Ran, F. Ann; Hsu, Patrick D. Cell, Vol. 156, Issue 5 https://doi.org/10.1016/j.cell.2014.02.001	journal	February 2014
Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System Xiao, Yibei; Luo, Min; Hayes, Robert P. Cell, Vol. 170, Issue 1 https://doi.org/10.1016/j.cell.2017.06.012	journal	June 2017
CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3 Westra, Edze R.; van Erp, Paul B. G.; Künne, Tim Molecular Cell, Vol. 46, Issue 5 https://doi.org/10.1016/j.molcel.2012.03.018	journal	June 2012
Two Distinct DNA Binding Modes Guide Dual Roles of a CRISPR-Cas Protein Complex Blosser, Timothy R.; Loeff, Luuk; Westra, Edze R. Molecular Cell, Vol. 58, Issue 1 https://doi.org/10.1016/j.molcel.2015.01.028	journal	April 2015
DNA Targeting by a Minimal CRISPR RNA-Guided Cascade Hochstrasser, Megan L.; Taylor, David W.; Kornfeld, Jack E. Molecular Cell, Vol. 63, Issue 5 https://doi.org/10.1016/j.molcel.2016.07.027	journal	September 2016
RecA: Regulation and Mechanism of a Molecular Search Engine Bell, Jason C.; Kowalczykowski, Stephen C. Trends in Biochemical Sciences, Vol. 41, Issue 6 https://doi.org/10.1016/j.tibs.2016.04.002	journal	June 2016
Determination of the extent of DNA bending by an adenine-thymine tract Koo, Hyeon Sook; Drak, Jacqueline; Rice, Janet A. Biochemistry, Vol. 29, Issue 17 https://doi.org/10.1021/bi00469a027	journal	May 1990
DNA bending at adenine · thymine tracts Koo, Hyeon-Sook; Wu, Hen-Ming; Crothers, Donald M. Nature, Vol. 320, Issue 6062 https://doi.org/10.1038/320501a0	journal	April 1986
Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA Bruner, Steven D.; Norman, Derek P. G.; Verdine, Gregory L. Nature, Vol. 403, Issue 6772 https://doi.org/10.1038/35002510	journal	February 2000
Crystal structure of a yeast TBP/TATA-box complex Kim, Youngchang; Geiger, James. H.; Hahn, Steven Nature, Vol. 365, Issue 6446 https://doi.org/10.1038/365512a0	journal	October 1993
Co-crystal structure of TBP recognizing the minor groove of a TATA element Kim, Joseph L.; Nikolov, Dimitar B.; Burley, Stephen K. Nature, Vol. 365, Issue 6446 https://doi.org/10.1038/365520a0	journal	October 1993
Structural basis for DNA bending by the architectural transcription factor LEF-1 Love, John J.; Li, Xiang; Case, David A. Nature, Vol. 376, Issue 6543 https://doi.org/10.1038/376791a0	journal	August 1995
A nucleotide-flipping mechanism from the structure of human uracil–DNA glycosylase bound to DNA Slupphaug, Geir; Mol, Clifford D.; Kavli, Bodil Nature, Vol. 384, Issue 6604 https://doi.org/10.1038/384087a0	journal	November 1996
Mechanism of homologous recombination from the RecA–ssDNA/dsDNA structures Chen, Zhucheng; Yang, Haijuan; Pavletich, Nikola P. Nature, Vol. 453, Issue 7194 https://doi.org/10.1038/nature06971	journal	May 2008
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 Sternberg, Samuel H.; Redding, Sy; Jinek, Martin Nature, Vol. 507, Issue 7490 https://doi.org/10.1038/nature13011	journal	January 2014
Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease Anders, Carolin; Niewoehner, Ole; Duerst, Alessia Nature, Vol. 513, Issue 7519 https://doi.org/10.1038/nature13579	journal	July 2014
The next generation of CRISPR–Cas technologies and applications Pickar-Oliver, Adrian; Gersbach, Charles A. Nature Reviews Molecular Cell Biology, Vol. 20, Issue 8 https://doi.org/10.1038/s41580-019-0131-5	journal	May 2019
Mechanism of strand exchange from RecA–DNA synaptic and D-loop structures Yang, Haijuan; Zhou, Chun; Dhar, Ankita Nature, Vol. 586, Issue 7831 https://doi.org/10.1038/s41586-020-2820-9	journal	October 2020
DNA stretching induces Cas9 off-target activity Newton, Matthew D.; Taylor, Benjamin J.; Driessen, Rosalie P. C. Nature Structural & Molecular Biology, Vol. 26, Issue 3 https://doi.org/10.1038/s41594-019-0188-z	journal	February 2019
Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes Szczelkun, Mark D.; Tikhomirova, Maria S.; Sinkunas, Tomas Proceedings of the National Academy of Sciences, Vol. 111, Issue 27 https://doi.org/10.1073/pnas.1402597111	journal	May 2014
Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin Proceedings of the National Academy of Sciences, Vol. 114, Issue 21 https://doi.org/10.1073/pnas.1619926114	journal	May 2017
Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling Ivanov, Ivan E.; Wright, Addison V.; Cofsky, Joshua C. Proceedings of the National Academy of Sciences, Vol. 117, Issue 11 https://doi.org/10.1073/pnas.1913445117	journal	March 2020
Energetic coupling between DNA bending and base pair opening. Ramstein, J.; Lavery, R. Proceedings of the National Academy of Sciences, Vol. 85, Issue 19 https://doi.org/10.1073/pnas.85.19.7231	journal	October 1988
Protein-induced bending and DNA cyclization. Kahn, J. D.; Crothers, D. M. Proceedings of the National Academy of Sciences, Vol. 89, Issue 14 https://doi.org/10.1073/pnas.89.14.6343	journal	July 1992
DNA Bending by EcoRI DNA Methyltransferase Accelerates Base Flipping but Compromises Specificity Allan, Barrett W.; Garcia, Ricardo; Maegley, Karen Journal of Biological Chemistry, Vol. 274, Issue 27 https://doi.org/10.1074/jbc.274.27.19269	journal	July 1999
Permanganate Oxidation Reactions of DNA: Perspective in Biological Studies Bui, Chinh T.; Rees, Kylee; Cotton, Richard G. H. Nucleosides, Nucleotides and Nucleic Acids, Vol. 22, Issue 9 https://doi.org/10.1081/NCN-120023276	journal	October 2003
Structure of a trapped endonuclease III-DNA covalent intermediate Fromme, J. C. The EMBO Journal, Vol. 22, Issue 13 https://doi.org/10.1093/emboj/cdg311	journal	July 2003
DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping Su, T. -J. Nucleic Acids Research, Vol. 33, Issue 10 https://doi.org/10.1093/nar/gki618	journal	June 2005
Revised UV extinction coefficients for nucleoside-5′-monophosphates and unpaired DNA and RNA Cavaluzzi, Michael J.; Borer, Philip N. Nucleic Acids Research, Vol. 32, Issue 1 https://doi.org/10.1093/nar/gnh015	journal	January 2004
DNA base repair – recognition and initiation of catalysis Dalhus, Bjørn; Laerdahl, Jon K.; Backe, Paul H. FEMS Microbiology Reviews, Vol. 33, Issue 6 https://doi.org/10.1111/j.1574-6976.2009.00188.x	journal	November 2009
CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes Barrangou, R.; Fremaux, C.; Deveau, H. Science, Vol. 315, Issue 5819 https://doi.org/10.1126/science.1138140	journal	March 2007
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Jinek, M.; Chylinski, K.; Fonfara, I. Science, Vol. 337, Issue 6096, p. 816-821 https://doi.org/10.1126/science.1225829	journal	June 2012
Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation Jinek, M.; Jiang, F.; Taylor, D. W. Science, Vol. 343, Issue 6176 https://doi.org/10.1126/science.1247997	journal	February 2014
Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees Schultz, S.; Shields, G.; Steitz, T. Science, Vol. 253, Issue 5023 https://doi.org/10.1126/science.1653449	journal	August 1991
A Cas9-guide RNA complex preorganized for target DNA recognition Jiang, F.; Zhou, K.; Ma, L. Science, Vol. 348, Issue 6242 https://doi.org/10.1126/science.aab1452	journal	June 2015
Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage Jiang, F.; Taylor, D. W.; Chen, J. S. Science, Vol. 351, Issue 6275 https://doi.org/10.1126/science.aad8282	journal	January 2016
Kinetics of dCas9 target search in Escherichia coli Jones, Daniel Lawson; Leroy, Prune; Unoson, Cecilia Science, Vol. 357, Issue 6358 https://doi.org/10.1126/science.aah7084	journal	September 2017
CRISPR–Cas9 Structures and Mechanisms Jiang, Fuguo; Doudna, Jennifer A. Annual Review of Biophysics, Vol. 46, Issue 1 https://doi.org/10.1146/annurev-biophys-062215-010822	journal	May 2017
Covalent Trapping of Protein-DNA Complexes Verdine, Gregory L.; Norman, Derek P. G. Annual Review of Biochemistry, Vol. 72, Issue 1 https://doi.org/10.1146/annurev.biochem.72.121801.161447	journal	June 2003
Real‐time observation of flexible domain movements in CRISPR–Cas9 Osuka, Saki; Isomura, Kazushi; Kajimoto, Shohei The EMBO Journal, Vol. 37, Issue 10 https://doi.org/10.15252/embj.201796941	journal	April 2018
CRISPR /Cas9 searches for a protospacer adjacent motif by lateral diffusion Globyte, Viktorija; Lee, Seung Hwan; Bae, Taegeun The EMBO Journal, Vol. 38, Issue 4 https://doi.org/10.15252/embj.201899466	journal	December 2018
Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION Nakane, Takanori; Kimanius, Dari; Lindahl, Erik eLife, Vol. 7 https://doi.org/10.7554/eLife.36861	journal	June 2018
CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks Cofsky, Joshua C.; Karandur, Deepti; Huang, Carolyn J. eLife, Vol. 9 https://doi.org/10.7554/eLife.55143	journal	June 2020

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