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

Singh, Digvijay; Sternberg, Samuel H.; Fei, Jingyi; Doudna, Jennifer A.; Ha, Taekjip

doi:10.1038/ncomms12778

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

Journal Article · Wed Sep 14 00:00:00 EDT 2016 · Nature Communications

DOI:https://doi.org/10.1038/ncomms12778· OSTI ID:1623849

Singh, Digvijay ^[1]; Sternberg, Samuel H. ^[2]; Fei, Jingyi ^[3]; Doudna, Jennifer A. ^[4];

^[5]

Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States). Dept. of Biophysics and Biophysical Chemistry, and Dept. of Biomedical Engineering; Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States). Center for Biophysics and Quantitative Biology
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
Univ. of Chicago, Chicago, IL (United States). Dept. of Biochemistry and Molecular Biology; Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States). Dept. of Physics and Center for the Physics of Living Cells; Howard Hughes Medical Institute, Baltimore, MD (United States)
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry and Dept. of Molecular and Cell Biology, an Innovative Genomics Initiative; Howard Hughes Medical Institute, Berkeley, CA (United States); Lawrence Berkeley National Lab (LBNL), Berkeley, CA (United States). Physical Biosciences Div.
Johns Hopkins Univ., School of Medicine, Baltimore, MD (United States). Dept. of Biophysics and Biophysical Chemistry; Johns Hopkins Univ., Baltimore, MD (United States). Dept. of Biophysics, and Dept. of Biomedical Engineering; Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States). Center for Biophysics and Quantitative Biology, Dept. of Physics, and Center for the Physics of Living Cells; Howard Hughes Medical Institute, Baltimore, 21205, Maryland, USA

Binding specificity of Cas9–guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9–RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s^-1 to >2 s^-1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s^-1), suggesting that extremely slow rejection could sequester Cas9–RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

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

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

Grant/Contract Number:: AC02-05CH11231; PHY-1430124; 1244557; GM065367; GM112659

OSTI ID:: 1623849

Journal Information:: Nature Communications, Vol. 7, Issue 1; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice Hu, Xixun; Meng, Xiangbing; Liu, Qing Plant Biotechnology Journal, Vol. 16, Issue 1 https://doi.org/10.1111/pbi.12771	journal	August 2017
The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation Zhang, Qian; Wen, Fengcai; Zhang, Siqi Science Advances, Vol. 5, Issue 11 https://doi.org/10.1126/sciadv.aaw9807	journal	November 2019
Core Hairpin Structure of SpCas9 sgRNA Functions in a Sequence- and Spatial Conformation–Dependent Manner Jiang, Mingjun; Ye, Yanzhen; Li, Juan SLAS TECHNOLOGY: Translating Life Sciences Innovation https://doi.org/10.1177/2472630320922813	journal	June 2020
Versatile transcription control based on reversible dCas9 binding Widom, Julia R.; Rai, Victoria; Rohlman, Christopher E. RNA, Vol. 25, Issue 11 https://doi.org/10.1261/rna.071613.119	journal	July 2019
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
A Low-Cost Time-Correlated Single Photon Counting Portable DNA Analyzer Tian, Yi; Wei, Liping; Ho, Derek Sensors, Vol. 19, Issue 13 https://doi.org/10.3390/s19132838	journal	June 2019
High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding Boyle, Evan A.; Andreasson, Johan O. L.; Chircus, Lauren M. Proceedings of the National Academy of Sciences, Vol. 114, Issue 21 https://doi.org/10.1073/pnas.1700557114	journal	May 2017
The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex Zeng, Yan; Cui, Yang; Zhang, Yong Nucleic Acids Research, Vol. 46, Issue 1 https://doi.org/10.1093/nar/gkx1117	journal	November 2017
ERASE: a novel surface reconditioning strategy for single-molecule experiments Broadwater Jr, D. W. Bo; Altman, Roger B.; Blanchard, Scott C. bioRxiv https://doi.org/10.1101/282632	posted_content	March 2018
Nuclease dead Cas9 is a programmable roadblock for DNA replication Whinn, Kelsey; Kaur, Gurleen; Lewis, Jacob S. https://doi.org/10.1101/455543	posted_content	October 2018
CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations Giulia, Palermo,; Yinglong, Miao,; C., Walker, Ross National Academy of Sciences https://doi.org/10.5167/uzh-138566	text	January 2017
Molecular Mechanism of Off-Target Effects in CRISPR-Cas9 Palermo, Giulia; Gravina Ricci, Clarisse; Chen, Janice S. Biophysical Journal, Vol. 116, Issue 3 https://doi.org/10.1016/j.bpj.2018.11.1731	journal	February 2019
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
Deciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamics Ricci, Clarisse G.; Chen, Janice S.; Miao, Yinglong ACS Central Science, Vol. 5, Issue 4 https://doi.org/10.1021/acscentsci.9b00020	journal	March 2019
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
Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease Rueda, Fernando Orden; Bista, Michal; Newton, Matthew D. Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/s41467-017-01732-9	journal	November 2017
Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a Swartjes, Thomas; Staals, Raymond H. J.; van der Oost, John Biochemical Society Transactions, Vol. 48, Issue 1 https://doi.org/10.1042/bst20190563	journal	December 2019
Game-Theoretical Modeling of Interviral Conflicts Mediated by Mini-CRISPR Arrays Iranzo, Jaime; Faure, Guilhem; Wolf, Yuri I. Frontiers in Microbiology, Vol. 11 https://doi.org/10.3389/fmicb.2020.00381	journal	March 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
GSSMD: New metric for robust and interpretable assay quality assessment and hit selection Park, Seongyong; Khan, Shujaat arXiv https://doi.org/10.48550/arxiv.2001.06384	preprint	January 2020

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