Deciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamics

Ricci, Clarisse G.; Chen, Janice S.; Miao, Yinglong; Jinek, Martin; Doudna, Jennifer A.; McCammon, J. Andrew; Palermo, Giulia

doi:10.1021/acscentsci.9b00020

Deciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamics

Journal Article · Thu Mar 07 00:00:00 EST 2019 · ACS Central Science

DOI:https://doi.org/10.1021/acscentsci.9b00020· OSTI ID:1642674

^[1]; Chen, Janice S. ^[2]; ^[3]; Jinek, Martin ^[4]; ^[2]; ^[1]; ^[5]

Univ. of California San Diego, La Jolla, CA (United States)
Univ. of California, Berkeley, CA (United States)
Univ. of Kansas, Lawrence, KS (United States)
Univ. of Zürich (Switzerland)
Univ. of California, Riverside, CA (United States)

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations-using Gaussian accelerated MD (GaMD)-are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a "lock" effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to "lock" the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to "lock" the catalytic HNH domain in an inactive "conformational checkpoint" is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the "locking" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: National Institute of General Medical Sciences; National Institutes of Health (NIH); National Science Foundation (NSF); USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1642674

Journal Information:: ACS Central Science, Journal Name: ACS Central Science Journal Issue: 4 Vol. 5; ISSN 2374-7943

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (48)

The Molecular Basis for Dual Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition Palermo, Giulia; Favia, Angelo D.; Convertino, Marino ChemMedChem, Vol. 11, Issue 12 https://doi.org/10.1002/cmdc.201500507	journal	November 2015
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
The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET Yang, Mengyi; Peng, Sijia; Sun, Ruirui Cell Reports, Vol. 22, Issue 2 https://doi.org/10.1016/j.celrep.2017.12.048	journal	January 2018
Structure and Dynamics of the CRISPR–Cas9 Catalytic Complex Palermo, Giulia Journal of Chemical Information and Modeling, Vol. 59, Issue 5 https://doi.org/10.1021/acs.jcim.8b00988	journal	February 2019
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
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
Refinement of the Cornell et al. Nucleic Acids Force Field Based on Reference Quantum Chemical Calculations of Glycosidic Torsion Profiles Zgarbová, Marie; Otyepka, Michal; Šponer, Jiří Journal of Chemical Theory and Computation, Vol. 7, Issue 9 https://doi.org/10.1021/ct200162x	journal	August 2011
Routine Access to Millisecond Time Scale Events with Accelerated Molecular Dynamics Pierce, Levi C. T.; Salomon-Ferrer, Romelia; Augusto F. de Oliveira, Cesar Journal of Chemical Theory and Computation, Vol. 8, Issue 9 https://doi.org/10.1021/ct300284c	journal	August 2012
Ion-water interaction potentials derived from free energy perturbation simulations Ȧqvist, Johan The Journal of Physical Chemistry, Vol. 94, Issue 21 https://doi.org/10.1021/j100384a009	journal	October 1990
Dynamics of B-DNA on the Microsecond Time Scale Pérez, Alberto; Luque, F. Javier; Orozco, Modesto Journal of the American Chemical Society, Vol. 129, Issue 47 https://doi.org/10.1021/ja0753546	journal	November 2007
Sharpening the Scissors: Mechanistic Details of CRISPR/Cas9 Improve Functional Understanding and Inspire Future Research Raper, Austin T.; Stephenson, Anthony A.; Suo, Zucai Journal of the American Chemical Society, Vol. 140, Issue 36 https://doi.org/10.1021/jacs.8b05469	journal	August 2018
Molecular Dynamics of DNA: Comparison of Force Fields and Terminal Nucleotide Definitions Ricci, Clarisse G.; de Andrade, Alex S. C.; Mottin, Melina The Journal of Physical Chemistry B, Vol. 114, Issue 30 https://doi.org/10.1021/jp1035663	journal	August 2010
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
High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells Fu, Yanfang; Foden, Jennifer A.; Khayter, Cyd Nature Biotechnology, Vol. 31, Issue 9 https://doi.org/10.1038/nbt.2623	journal	June 2013
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
A highly specific SpCas9 variant is identified by in vivo screening in yeast Casini, Antonio; Olivieri, Michele; Petris, Gianluca Nature Biotechnology, Vol. 36, Issue 3 https://doi.org/10.1038/nbt.4066	journal	January 2018
Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Shibata, Mikihiro; Nishimasu, Hiroshi; Kodera, Noriyuki Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/s41467-017-01466-8	journal	November 2017
The chemistry of Cas9 and its CRISPR colleagues Chen, Janice S.; Doudna, Jennifer A. Nature Reviews Chemistry, Vol. 1, Issue 10 https://doi.org/10.1038/s41570-017-0078	journal	October 2017
Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis Singh, Digvijay; Wang, Yanbo; Mallon, John Nature Structural & Molecular Biology, Vol. 25, Issue 4 https://doi.org/10.1038/s41594-018-0051-7	journal	April 2018
Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations Zuo, Zhicheng; Liu, Jin Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep37584	journal	November 2016
Accelerated molecular dynamics: A promising and efficient simulation method for biomolecules Hamelberg, Donald; Mongan, John; McCammon, J. Andrew The Journal of Chemical Physics, Vol. 120, Issue 24 https://doi.org/10.1063/1.1755656	journal	June 2004
Molecular dynamics with coupling to an external bath Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F. The Journal of Chemical Physics, Vol. 81, Issue 8 https://doi.org/10.1063/1.448118	journal	October 1984
Graded activation and free energy landscapes of a muscarinic G-protein–coupled receptor Miao, Yinglong; McCammon, J. Andrew Proceedings of the National Academy of Sciences, Vol. 113, Issue 43 https://doi.org/10.1073/pnas.1614538113	journal	October 2016
Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor Miao, Yinglong; McCammon, J. Andrew Proceedings of the National Academy of Sciences, Vol. 115, Issue 12 https://doi.org/10.1073/pnas.1800756115	journal	March 2018
Unconstrained enhanced sampling for free energy calculations of biomolecules: a review Miao, Yinglong; McCammon, J. Andrew Molecular Simulation, Vol. 42, Issue 13 https://doi.org/10.1080/08927022.2015.1121541	journal	July 2016
Molecular dynamics of a κB DNA element: base flipping via cross-strand intercalative stacking in a microsecond-scale simulation Mura, Cameron; McCammon, J. Andrew Nucleic Acids Research, Vol. 36, Issue 15 https://doi.org/10.1093/nar/gkn473	journal	July 2008
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
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
Refinement of the AMBER Force Field for Nucleic Acids: Improving the Description of α/γ Conformers Pérez, Alberto; Marchán, Iván; Svozil, Daniel Biophysical Journal, Vol. 92, Issue 11 https://doi.org/10.1529/biophysj.106.097782	journal	June 2007
Key role of the REC lobe during CRISPR–Cas9 activation by ‘sensing’, ‘regulating’, and ‘locking’ the catalytic HNH domain Palermo, Giulia; Chen, Janice S.; Ricci, Clarisse G. Quarterly Reviews of Biophysics, Vol. 51 https://doi.org/10.1017/S0033583518000070	journal	January 2018
Gaussian Accelerated Molecular Dynamics: Unconstrained Enhanced Sampling and Free Energy Calculation Miao, Yinglong; Feher, Victoria A.; McCammon, J. Andrew Journal of Chemical Theory and Computation, Vol. 11, Issue 8 https://doi.org/10.1021/acs.jctc.5b00436	journal	July 2015
Development of Site-Specific Mg ²⁺ –RNA Force Field Parameters: A Dream or Reality? Guidelines from Combined Molecular Dynamics and Quantum Mechanics Simulations Casalino, Lorenzo; Palermo, Giulia; Abdurakhmonova, Nodira Journal of Chemical Theory and Computation, Vol. 13, Issue 1 https://doi.org/10.1021/acs.jctc.6b00905	journal	December 2016
Performance of Molecular Mechanics Force Fields for RNA Simulations: Stability of UUCG and GNRA Hairpins Banáš, Pavel; Hollas, Daniel; Zgarbová, Marie Journal of Chemical Theory and Computation, Vol. 6, Issue 12 https://doi.org/10.1021/ct100481h	journal	November 2010
Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9 Palermo, Giulia; Ricci, Clarisse G.; Fernando, Amendra Journal of the American Chemical Society, Vol. 139, Issue 45 https://doi.org/10.1021/jacs.7b05313	journal	August 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
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
Enhanced proofreading governs CRISPR–Cas9 targeting accuracy Chen, Janice S.; Dagdas, Yavuz S.; Kleinstiver, Benjamin P. Nature, Vol. 550, Issue 7676 https://doi.org/10.1038/nature24268	journal	September 2017
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
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
Brownian dynamics: Its application to ionic solutions Turq, Pierre; Lantelme, Frédéric; Friedman, Harold L. The Journal of Chemical Physics, Vol. 66, Issue 7 https://doi.org/10.1063/1.434317	journal	April 1977
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
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
Towards a molecular dynamics consensus view of B-DNA flexibility Pérez, Alberto; Lankas, Filip; Luque, F. Javier Nucleic Acids Research, Vol. 36, Issue 7 https://doi.org/10.1093/nar/gkn082	journal	February 2008
Conformational analysis of nucleic acids revisited: Curves+ Lavery, R.; Moakher, M.; Maddocks, J. H. Nucleic Acids Research, Vol. 37, Issue 17 https://doi.org/10.1093/nar/gkp608	journal	July 2009
A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9 Dagdas, Yavuz S.; Chen, Janice S.; Sternberg, Samuel H. Science Advances, Vol. 3, Issue 8 https://doi.org/10.1126/sciadv.aao0027	journal	August 2017
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
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
Amber 16, University of California, San Francisco. Case, David A.; Betz, Robin M.; Cerutti, D. S. University of California, San Francisco. https://doi.org/10.13140/rg.2.2.27958.70729	text	January 2016

Cited By (9)

Mechanisms of γ-Secretase Activation and Substrate Processing Bhattarai, Apurba; Devkota, Sujan; Bhattarai, Sanjay ACS Central Science, Vol. 6, Issue 6 https://doi.org/10.1021/acscentsci.0c00296	journal	June 2020
Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9 Mitchell, Brandon P.; Hsu, Rohaine V.; Medrano, Marco A. Frontiers in Molecular Biosciences, Vol. 7 https://doi.org/10.3389/fmolb.2020.00039	journal	March 2020
Disclosing the Impact of Carcinogenic SF3b Mutations on Pre-mRNA Recognition Via All-Atom Simulations Borišek, Jure; Saltalamacchia, Andrea; Gallì, Anna Biomolecules, Vol. 9, Issue 10 https://doi.org/10.3390/biom9100633	journal	October 2019
Pathway and mechanism of drug binding to chemokine receptors revealed by accelerated molecular simulations Pawnikar, Shristi; Miao, Yinglong Future Medicinal Chemistry, Vol. 12, Issue 13 https://doi.org/10.4155/fmc-2020-0044	journal	July 2020
CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects Manghwar, Hakim; Li, Bo; Ding, Xiao Advanced Science, Vol. 7, Issue 6 https://doi.org/10.1002/advs.201902312	journal	March 2020
G‐Protein‐Coupled Receptor–Membrane Interactions Depend on the Receptor Activation State Bhattarai, Apurba; Wang, Jinan; Miao, Yinglong Journal of Computational Chemistry, Vol. 41, Issue 5 https://doi.org/10.1002/jcc.26082	journal	October 2019
NMR and computational methods for molecular resolution of allosteric pathways in enzyme complexes East, Kyle W.; Skeens, Erin; Cui, Jennifer Y. Biophysical Reviews, Vol. 12, Issue 1 https://doi.org/10.1007/s12551-019-00609-z	journal	December 2019
Conformational flexibility correlates with glucose tolerance for point mutations in β-glucosidases – a computational study Lima, Leonardo Henrique Franca de; Fernandez-Quintéro, Monica Lisa; Rocha, Rafael Eduardo Oliveira Journal of Biomolecular Structure and Dynamics https://doi.org/10.1080/07391102.2020.1734484	journal	March 2020
Conformational flexibility correlates with glucose tolerance for point mutations in β-glucosidases – a computational study Lima, Leonardo Henrique Franca De; Fernandez-Quintéro, Monica Lisa; Rocha, Rafael Eduardo Oliveira Taylor & Francis https://doi.org/10.6084/m9.figshare.11913315.v2	text	January 2020

Similar Records

Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

Journal Article · Tue Aug 01 20:00:00 EDT 2017 · Journal of the American Chemical Society · OSTI ID:1489021

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

Journal Article · Tue Sep 13 20:00:00 EDT 2016 · Nature Communications · OSTI ID:1623849

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

Journal Article · Mon Jul 31 20:00:00 EDT 2017 · Science Advances · OSTI ID:1625977

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
crystal cleavage
genetics
genomics
nucleic acid structure
polyacrylamide

Deciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamics

Citation Formats

References (48)

Cited By (9)

Similar Records

Related Subjects