Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

Palermo, Giulia; Ricci, Clarisse G.; Fernando, Amendra; Basak, Rajshekhar; Jinek, Martin; Rivalta, Ivan; Batista, Victor S.; McCammon, J. Andrew

doi:10.1021/jacs.7b05313

Title: Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

Journal Article · Wed Aug 02 00:00:00 EDT 2017 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/jacs.7b05313· OSTI ID:1489021

^[1];

^[2]; Basak, Rajshekhar ^[2]; Jinek, Martin ^[3];

^[4];

^[2]; McCammon, J. Andrew ^[1]

Univ. of California San Diego, La Jolla, CA (United States)
Yale Univ., New Haven, CT (United States)
Univ. of Zurich, Zurich (Switzerland)
Univ. Claude Bernard Lyon, Lyon (France)

CRISPR-Cas9 is a genome editing technology with major impact in life sciences. In this system, the endonuclease Cas9 generates double strand breaks in DNA upon RNA-guided recognition of a complementary DNA sequence, which strictly requires the presence of a protospacer adjacent motif (PAM) next to the target site. Although PAM recognition is essential for cleavage, it is unknown whether and how PAM binding activates Cas9 for DNA cleavage at spatially distant sites. Here, we find evidence of a PAM-induced allosteric mechanism revealed by microsecond molecular dynamics simulations. PAM acts as an allosteric effector and triggers the interdependent conformational dynamics of the Cas9 catalytic domains (HNH and RuvC), responsible for concerted cleavage of the two DNA strands. As a result, targeting such an allosteric mechanism should enable control of CRISPR-Cas9 functionality.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)

Sponsoring Organization:: USDOE

OSTI ID:: 1489021

Journal Information:: Journal of the American Chemical Society, Vol. 139, Issue 45; ISSN 0002-7863

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 73 works

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References (22)

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
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
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
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
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
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
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
Catalytic Metal Ions and Enzymatic Processing of DNA and RNA Palermo, Giulia; Cavalli, Andrea; Klein, Michael L. Accounts of Chemical Research, Vol. 48, Issue 2 https://doi.org/10.1021/ar500314j	journal	January 2015
CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations Palermo, Giulia; Miao, Yinglong; Walker, Ross C. Proceedings of the National Academy of Sciences, Vol. 114, Issue 28 https://doi.org/10.1073/pnas.1707645114	journal	June 2017
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
Generalized correlation for biomolecular dynamics Lange, Oliver F.; Grubmüller, Helmut Proteins: Structure, Function, and Bioinformatics, Vol. 62, Issue 4 https://doi.org/10.1002/prot.20784	journal	December 2005
Protein Allostery and Conformational Dynamics Guo, Jingjing; Zhou, Huan-Xiang Chemical Reviews, Vol. 116, Issue 11 https://doi.org/10.1021/acs.chemrev.5b00590	journal	February 2016
Allosteric cross-talk in chromatin can mediate drug-drug synergy Adhireksan, Zenita; Palermo, Giulia; Riedel, Tina Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms14860	journal	March 2017
Dynamical networks in tRNA:protein complexes Sethi, A.; Eargle, J.; Black, A. A. Proceedings of the National Academy of Sciences, Vol. 106, Issue 16 https://doi.org/10.1073/pnas.0810961106	journal	April 2009
Allosteric pathways in imidazole glycerol phosphate synthase Rivalta, I.; Sultan, M. M.; Lee, N. -S. Proceedings of the National Academy of Sciences, Vol. 109, Issue 22 https://doi.org/10.1073/pnas.1120536109	journal	May 2012
Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep19940	journal	January 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
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
Engineered CRISPR-Cas9 nucleases with altered PAM specificities Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q. Nature, Vol. 523, Issue 7561 https://doi.org/10.1038/nature14592	journal	June 2015
Electrostatics of nanosystems: Application to microtubules and the ribosome Baker, N. A.; Sept, D.; Joseph, S. Proceedings of the National Academy of Sciences, Vol. 98, Issue 18, p. 10037-10041 https://doi.org/10.1073/pnas.181342398	journal	August 2001
Improvement of Soybean; A Way Forward Transition from Genetic Engineering to New Plant Breeding Technologies Rahman, Saleem Ur; McCoy, Evan; Raza, Ghulam Molecular Biotechnology, Vol. 65, Issue 2 https://doi.org/10.1007/s12033-022-00456-6	journal	February 2022
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

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PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures Gleditzsch, Daniel; Pausch, Patrick; Müller-Esparza, Hanna RNA Biology, Vol. 16, Issue 4 https://doi.org/10.1080/15476286.2018.1504546	journal	September 2018
Allostery in Its Many Disguises: From Theory to Applications Wodak, Shoshana J.; Paci, Emanuele; Dokholyan, Nikolay V. Structure, Vol. 27, Issue 4 https://doi.org/10.1016/j.str.2019.01.003	journal	April 2019
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