A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

Dagdas, Yavuz S.; Chen, Janice S.; Sternberg, Samuel H.; Doudna, Jennifer A.; Yildiz, Ahmet

doi:10.1126/sciadv.aao0027

Title: A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

Journal Article · 01 August 2017 · Science Advances

DOI: https://doi.org/10.1126/sciadv.aao0027 · OSTI ID:1625977

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Univ. of California, Berkeley, CA (United States). Biophysics Graduate Group
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Biosciences Division
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). Dept. of Physics

The Cas9 endonuclease is widely used for genome engineering applications by programming its single-guide RNA, and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer, we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

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

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1625977

Journal Information:: Science Advances, Vol. 3, Issue 8; ISSN 2375-2548

Publisher:: AAASCopyright Statement

Country of Publication:: United States

Language:: English

References (60)

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
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
Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations Palermo, Giulia; Miao, Yinglong; Walker, Ross C. ACS Central Science, Vol. 2, Issue 10 https://doi.org/10.1021/acscentsci.6b00218	journal	September 2016
The new frontier of genome engineering with CRISPR-Cas9 Doudna, Jennifer A.; Charpentier, Emmanuelle Science, Vol. 346, Issue 6213 https://doi.org/10.1126/science.1258096	journal	November 2014
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria Gasiunas, G.; Barrangou, R.; Horvath, P. Proceedings of the National Academy of Sciences, Vol. 109, Issue 39, p. E2579-E2586 https://doi.org/10.1073/pnas.1208507109	journal	September 2012
Expanding the Biologist’s Toolkit with CRISPR-Cas9 Sternberg, Samuel H.; Doudna, Jennifer A. Molecular Cell, Vol. 58, Issue 4 https://doi.org/10.1016/j.molcel.2015.02.032	journal	May 2015
Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering Wright, Addison V.; Nuñez, James K.; Doudna, Jennifer A. Cell, Vol. 164, Issue 1-2 https://doi.org/10.1016/j.cell.2015.12.035	journal	January 2016
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
Conformational control of DNA target cleavage by CRISPR–Cas9 Sternberg, Samuel H.; LaFrance, Benjamin; Kaplan, Matias Nature, Vol. 527, Issue 7576 https://doi.org/10.1038/nature15544	journal	October 2015
Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9 Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro Molecular Cell, Vol. 61, Issue 6 https://doi.org/10.1016/j.molcel.2016.02.018	journal	March 2016
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
Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy Gori, Jennifer L.; Hsu, Patrick D.; Maeder, Morgan L. Human Gene Therapy, Vol. 26, Issue 7 https://doi.org/10.1089/hum.2015.074	journal	July 2015
Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9 Singh, Digvijay; Sternberg, Samuel H.; Fei, Jingyi Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms12778	journal	September 2016
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
Target specificity of the CRISPR-Cas9 system Wu, Xuebing; Kriz, Andrea J.; Sharp, Phillip A. Quantitative Biology, Vol. 2, Issue 2 https://doi.org/10.1007/s40484-014-0030-x	journal	June 2014
G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding Ray, S.; Bandaria, J. N.; Qureshi, M. H. Proceedings of the National Academy of Sciences, Vol. 111, Issue 8 https://doi.org/10.1073/pnas.1321436111	journal	February 2014
Single-Molecule Studies of Synaptotagmin and Complexin Binding to the SNARE Complex Bowen, Mark E.; Weninger, Keith; Ernst, James Biophysical Journal, Vol. 89, Issue 1 https://doi.org/10.1529/biophysj.104.054064	journal	July 2005
Unravelling the structural and mechanistic basis of CRISPR–Cas systems van der Oost, John; Westra, Edze R.; Jackson, Ryan N. Nature Reviews Microbiology, Vol. 12, Issue 7 https://doi.org/10.1038/nrmicro3279	journal	June 2014
Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9 Anders, Carolin; Bargsten, Katja; Jinek, Martin Molecular Cell, Vol. 61, Issue 6 https://doi.org/10.1016/j.molcel.2016.02.020	journal	March 2016
Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA Richardson, Christopher D.; Ray, Graham J.; DeWitt, Mark A. Nature Biotechnology, Vol. 34, Issue 3 https://doi.org/10.1038/nbt.3481	journal	January 2016
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
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
Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease Lim, Youngbin; Bak, So Young; Sung, Keewon Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms13350	journal	November 2016
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases Tsai, Shengdar Q.; Zheng, Zongli; Nguyen, Nhu T. Nature Biotechnology, Vol. 33, Issue 2 https://doi.org/10.1038/nbt.3117	journal	December 2014
Development and Applications of CRISPR-Cas9 for Genome Engineering Hsu, Patrick D.; Lander, Eric S.; Zhang, Feng Cell, Vol. 157, Issue 6 https://doi.org/10.1016/j.cell.2014.05.010	journal	June 2014
Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems Mohanraju, Prarthana; Makarova, Kira S.; Zetsche, Bernd Science, Vol. 353, Issue 6299 https://doi.org/10.1126/science.aad5147	journal	August 2016
High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S. Nature, Vol. 529, Issue 7587 https://doi.org/10.1038/nature16526	journal	January 2016
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells Wu, Xuebing; Scott, David A.; Kriz, Andrea J. Nature Biotechnology, Vol. 32, Issue 7 https://doi.org/10.1038/nbt.2889	journal	April 2014
CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity Barrangou, Rodolphe; Marraffini, Luciano A. Molecular Cell, Vol. 54, Issue 2 https://doi.org/10.1016/j.molcel.2014.03.011	journal	April 2014
Rationally engineered Cas9 nucleases with improved specificity Slaymaker, I. M.; Gao, L.; Zetsche, B. Science, Vol. 351, Issue 6268 https://doi.org/10.1126/science.aad5227	journal	December 2015
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs Fu, Yanfang; Sander, Jeffry D.; Reyon, Deepak Nature Biotechnology, Vol. 32, Issue 3 https://doi.org/10.1038/nbt.2808	journal	January 2014
DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9 Sternberg, Samuel H.; Redding, Sy; Jinek, Martin Biophysical Journal, Vol. 106, Issue 2 https://doi.org/10.1016/j.bpj.2013.11.3848	journal	January 2014
Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain Alić, Ivan; Goh, Pollyanna A.; Murray, Aoife Molecular Psychiatry https://doi.org/10.1038/s41380-020-0806-5	journal	July 2020
Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9 Anders, Carolin; Bargsten, Katja; Jinek, Martin Cell Press (Elsevier) https://doi.org/10.5167/uzh-125049	text	January 2016
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 Sternberg, Samuel H.; Redding, Sy; Jinek, Martin Nature Publishing Group https://doi.org/10.5167/uzh-96985	text	January 2014
Structures of Cas9 endonucleases reveal RNA-mediated conformational activation Jinek, Martin; Jiang, Fuguo; Taylor, David W. American Association for the Advancement of Science https://doi.org/10.5167/uzh-96990	text	January 2014
Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations Palermo, Giulia; Miao, Yinglong; Walker, Ross C. American Chemical Society (ACS) https://doi.org/10.5167/uzh-127230	text	January 2016
Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease Carolin, Anders,; Ole, Niewoehner,; Alessia, Duerst, Nature Publishing Group https://doi.org/10.5167/uzh-99826	text	January 2014
Target specificity of the CRISPR-Cas9 system Wu, Xuebing; Kriz, Andrea J.; Sharp, Phillip A. Quantitative Biology, Vol. 2, Issue 2 https://doi.org/10.1007/s40484-014-0030-x	journal	June 2014
DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9 Sternberg, Samuel H.; Redding, Sy; Jinek, Martin Biophysical Journal, Vol. 106, Issue 2 https://doi.org/10.1016/j.bpj.2013.11.3848	journal	January 2014
Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering Wright, Addison V.; Nuñez, James K.; Doudna, Jennifer A. Cell, Vol. 164, Issue 1-2 https://doi.org/10.1016/j.cell.2015.12.035	journal	January 2016
CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity Barrangou, Rodolphe; Marraffini, Luciano A. Molecular Cell, Vol. 54, Issue 2 https://doi.org/10.1016/j.molcel.2014.03.011	journal	April 2014
Expanding the Biologist’s Toolkit with CRISPR-Cas9 Sternberg, Samuel H.; Doudna, Jennifer A. Molecular Cell, Vol. 58, Issue 4 https://doi.org/10.1016/j.molcel.2015.02.032	journal	May 2015
Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9 Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro Molecular Cell, Vol. 61, Issue 6 https://doi.org/10.1016/j.molcel.2016.02.018	journal	March 2016
Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9 Anders, Carolin; Bargsten, Katja; Jinek, Martin Molecular Cell, Vol. 61, Issue 6 https://doi.org/10.1016/j.molcel.2016.02.020	journal	March 2016
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
High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S. Nature, Vol. 529, Issue 7587 https://doi.org/10.1038/nature16526	journal	January 2016
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells Wu, Xuebing; Scott, David A.; Kriz, Andrea J. Nature Biotechnology, Vol. 32, Issue 7 https://doi.org/10.1038/nbt.2889	journal	April 2014
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases Tsai, Shengdar Q.; Zheng, Zongli; Nguyen, Nhu T. Nature Biotechnology, Vol. 33, Issue 2 https://doi.org/10.1038/nbt.3117	journal	December 2014
Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease Lim, Youngbin; Bak, So Young; Sung, Keewon Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms13350	journal	November 2016
Unravelling the structural and mechanistic basis of CRISPR–Cas systems van der Oost, John; Westra, Edze R.; Jackson, Ryan N. Nature Reviews Microbiology, Vol. 12, Issue 7 https://doi.org/10.1038/nrmicro3279	journal	June 2014
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria Gasiunas, G.; Barrangou, R.; Horvath, P. Proceedings of the National Academy of Sciences, Vol. 109, Issue 39, p. E2579-E2586 https://doi.org/10.1073/pnas.1208507109	journal	September 2012
G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding Ray, S.; Bandaria, J. N.; Qureshi, M. H. Proceedings of the National Academy of Sciences, Vol. 111, Issue 8 https://doi.org/10.1073/pnas.1321436111	journal	February 2014
Fulfilling the dream of a perfect genome editing tool Spies, Maria Proceedings of the National Academy of Sciences, Vol. 111, Issue 28 https://doi.org/10.1073/pnas.1408985111	journal	July 2014
Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy Gori, Jennifer L.; Hsu, Patrick D.; Maeder, Morgan L. Human Gene Therapy, Vol. 26, Issue 7 https://doi.org/10.1089/hum.2015.074	journal	July 2015
High-throughput biochemical profiling reveals Cas9 off-target binding and unbinding heterogeneity Boyle, Evan A.; Andreasson, Johan O. L.; Chircus, Lauren M. https://doi.org/10.1101/059782	posted_content	June 2016
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
Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems Mohanraju, Prarthana; Makarova, Kira S.; Zetsche, Bernd Science, Vol. 353, Issue 6299 https://doi.org/10.1126/science.aad5147	journal	August 2016
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
Single-Molecule Studies of Synaptotagmin and Complexin Binding to the SNARE Complex Bowen, Mark E.; Weninger, Keith; Ernst, James Biophysical Journal, Vol. 89, Issue 1 https://doi.org/10.1529/biophysj.104.054064	journal	July 2005

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