Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.; Schoeniger, Joseph S.; Harmon, Brooke Nicole

doi:10.1021/acs.analchem.8b01155

Title: Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

Journal Article · Mon May 14 00:00:00 EDT 2018 · Analytical Chemistry

DOI:https://doi.org/10.1021/acs.analchem.8b01155· OSTI ID:1457411

Seamon, Kyle Jeffrey ^[1]; Light, Yooli Kim ^[1]; Saada, Edwin A. ^[1]; Schoeniger, Joseph S. ^[1];

^[1]

Sandia National Lab. (SNL-CA), Livermore, CA (United States)

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

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Research Organization:: Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

Sponsoring Organization:: USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: AC04-94AL85000

OSTI ID:: 1457411

Report Number(s):: SAND-2018-2676J; 661465

Journal Information:: Analytical Chemistry, Vol. 90, Issue 11; ISSN 0003-2700

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 14 works

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References (64)

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 targeting specificity of RNA-guided Cas9 nucleases Hsu, Patrick D.; Scott, David A.; Weinstein, Joshua A. Nature Biotechnology, Vol. 31, Issue 9, p. 827-832 https://doi.org/10.1038/nbt.2647	journal	July 2013
High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity Pattanayak, Vikram; Lin, Steven; Guilinger, John P. Nature Biotechnology, Vol. 31, Issue 9 https://doi.org/10.1038/nbt.2673	journal	August 2013
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
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
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
One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering Wang, Haoyi; Yang, Hui; Shivalila, Chikdu S. Cell, Vol. 153, Issue 4, p. 910-918 https://doi.org/10.1016/j.cell.2013.04.025	journal	May 2013
Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease Gratz, Scott J.; Cummings, Alexander M.; Nguyen, Jennifer N. Genetics, Vol. 194, Issue 4 https://doi.org/10.1534/genetics.113.152710	journal	May 2013
Efficient genome editing in zebrafish using a CRISPR-Cas system Hwang, Woong Y.; Fu, Yanfang; Reyon, Deepak Nature Biotechnology, Vol. 31, Issue 3, p. 227-229 https://doi.org/10.1038/nbt.2501	journal	January 2013
Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis: CRISPR-Cas Genome Editing in Xenopus Nakayama, Takuya; Fish, Margaret B.; Fisher, Marilyn genesis, Vol. 51, Issue 12 https://doi.org/10.1002/dvg.22720	journal	November 2013
Multiplex Genome Engineering Using CRISPR/Cas Systems Cong, L.; Ran, F. A.; Cox, D. Science, Vol. 339, Issue 6121, p. 819-823 https://doi.org/10.1126/science.1231143	journal	January 2013
RNA-Guided Human Genome Engineering via Cas9 Mali, P.; Yang, L.; Esvelt, K. M. Science, Vol. 339, Issue 6121, p. 823-826 https://doi.org/10.1126/science.1232033	journal	January 2013
CRISPR/Cas9 – Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms Yao, Xuan; Wang, Xing; Liu, Junlai EBioMedicine, Vol. 20 https://doi.org/10.1016/j.ebiom.2017.05.015	journal	June 2017
Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA Long, C.; McAnally, J. R.; Shelton, J. M. Science, Vol. 345, Issue 6201 https://doi.org/10.1126/science.1254445	journal	August 2014
In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy Nelson, C. E.; Hakim, C. H.; Ousterout, D. G. Science, Vol. 351, Issue 6271 https://doi.org/10.1126/science.aad5143	journal	December 2015
In vivo gene editing in dystrophic mouse muscle and muscle stem cells Tabebordbar, M.; Zhu, K.; Cheng, J. K. W. Science, Vol. 351, Issue 6271 https://doi.org/10.1126/science.aad5177	journal	December 2015
Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9 Wu, Yuxuan; Liang, Dan; Wang, Yinghua Cell Stem Cell, Vol. 13, Issue 6 https://doi.org/10.1016/j.stem.2013.10.016	journal	December 2013
Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing Ding, Qiurong; Strong, Alanna; Patel, Kevin M. Circulation Research, Vol. 115, Issue 5 https://doi.org/10.1161/CIRCRESAHA.115.304351	journal	August 2014
A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice Yang, Yang; Wang, Lili; Bell, Peter Nature Biotechnology, Vol. 34, Issue 3 https://doi.org/10.1038/nbt.3469	journal	February 2016
Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase Cinesi, Cinzia; Aeschbach, Lorène; Yang, Bin Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms13272	journal	November 2016
Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9 Pinto, Belinda S.; Saxena, Tanvi; Oliveira, Ruan Molecular Cell, Vol. 68, Issue 3 https://doi.org/10.1016/j.molcel.2017.09.033	journal	November 2017
In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration Suzuki, Keiichiro; Tsunekawa, Yuji; Hernandez-Benitez, Reyna Nature, Vol. 540, Issue 7631 https://doi.org/10.1038/nature20565	journal	November 2016
Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression Koo, Taeyoung; Yoon, A-Rum; Cho, Hee-Yeon Nucleic Acids Research, Vol. 45, Issue 13 https://doi.org/10.1093/nar/gkx490	journal	May 2017
CRISPR/Cas9-mediated genome editing via postnatal administration of AAV vector cures haemophilia B mice Ohmori, Tsukasa; Nagao, Yasumitsu; Mizukami, Hiroaki Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-04625-5	journal	June 2017
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity Hu, Johnny H.; Miller, Shannon M.; Geurts, Maarten H. Nature, Vol. 556, Issue 7699 https://doi.org/10.1038/nature26155	journal	February 2018
In vivo genome editing using Staphylococcus aureus Cas9 Ran, F. Ann; Cong, Le; Yan, Winston X. Nature, Vol. 520, Issue 7546 https://doi.org/10.1038/nature14299	journal	April 2015
In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni Kim, Eunji; Koo, Taeyoung; Park, Sung Wook Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms14500	journal	February 2017
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
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 Doench, John G.; Fusi, Nicolo; Sullender, Meagan Nature Biotechnology, Vol. 34, Issue 2 https://doi.org/10.1038/nbt.3437	journal	January 2016
Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency Zhang, Jian-Ping; Li, Xiao-Lan; Neises, Amanda Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep28566	journal	June 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
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
Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage Kulcsár, Péter István; Tálas, András; Huszár, Krisztina Genome Biology, Vol. 18, Issue 1 https://doi.org/10.1186/s13059-017-1318-8	journal	October 2017
Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization Senturk, Serif; Shirole, Nitin H.; Nowak, Dawid G. Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms14370	journal	February 2017
Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos Tu, Zhuchi; Yang, Weili; Yan, Sen Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/srep42081	journal	February 2017
Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch Oakes, Benjamin L.; Nadler, Dana C.; Flamholz, Avi Nature Biotechnology, Vol. 34, Issue 6 https://doi.org/10.1038/nbt.3528	journal	May 2016
A chemical-inducible CRISPR–Cas9 system for rapid control of genome editing Liu, Kaiwen Ivy; Ramli, Muhammad Nadzim Bin; Woo, Cheok Wei Ariel Nature Chemical Biology, Vol. 12, Issue 11 https://doi.org/10.1038/nchembio.2179	journal	September 2016
Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics Rose, John C.; Stephany, Jason J.; Valente, William J. Nature Methods, Vol. 14, Issue 9 https://doi.org/10.1038/nmeth.4368	journal	July 2017
Disabling Cas9 by an anti-CRISPR DNA mimic Shin, Jiyung; Jiang, Fuguo; Liu, Jun-Jie Science Advances, Vol. 3, Issue 7 https://doi.org/10.1126/sciadv.1701620	journal	July 2017
Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA Mekler, Vladimir; Minakhin, Leonid; Semenova, Ekaterina Nucleic Acids Research, Vol. 44, Issue 6 https://doi.org/10.1093/nar/gkw138	journal	March 2016
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 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification Zhang, Kaixiang; Deng, Ruijie; Li, Yue Chemical Science, Vol. 7, Issue 8 https://doi.org/10.1039/C6SC01355D	journal	January 2016
Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying Analytical Chemistry, Vol. 90, Issue 3 https://doi.org/10.1021/acs.analchem.7b04542	journal	January 2018
In Vitro Evaluation of CRISPR/Cas9 Function by an Electrochemiluminescent Assay Liu, Weipeng; Yu, Huiheng; Zhou, Xiaoming Analytical Chemistry, Vol. 88, Issue 17 https://doi.org/10.1021/acs.analchem.6b02338	journal	August 2016
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
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q. Nature Biotechnology, Vol. 33, Issue 12 https://doi.org/10.1038/nbt.3404	journal	November 2015
RNA-dependent RNA targeting by CRISPR-Cas9 Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine eLife, Vol. 7 https://doi.org/10.7554/eLife.32724	journal	January 2018
Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo Zuris, John A.; Thompson, David B.; Shu, Yilai Nature Biotechnology, Vol. 33, Issue 1 https://doi.org/10.1038/nbt.3081	journal	October 2014
NIH Image to ImageJ: 25 years of image analysis Schneider, Caroline A.; Rasband, Wayne S.; Eliceiri, Kevin W. Nature Methods, Vol. 9, Issue 7 https://doi.org/10.1038/nmeth.2089	journal	June 2012
Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II Xia, Hongyan; Gravelsina, Sabine; Öhrmalm, Christina BioTechniques, Vol. 60, Issue 1 https://doi.org/10.2144/000114370	journal	January 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
Why Hofmeister effects of many salts favor protein folding but not DNA helix formation Pegram, Laurel M.; Wendorff, Timothy; Erdmann, Robert Proceedings of the National Academy of Sciences, Vol. 107, Issue 17 https://doi.org/10.1073/pnas.0913376107	journal	April 2010
Potent Inhibition of Human Apurinic/Apyrimidinic Endonuclease 1 by Arylstibonic Acids Seiple, Lauren A.; Cardellina, John H.; Akee, Rhone Molecular Pharmacology, Vol. 73, Issue 3 https://doi.org/10.1124/mol.107.042622	journal	November 2007
Effect of Genome Size on AAV Vector Packaging Wu, Zhijian; Yang, Hongyan; Colosi, Peter Molecular Therapy, Vol. 18, Issue 1 https://doi.org/10.1038/mt.2009.255	journal	January 2010
CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9 Dugar, Gaurav; Leenay, Ryan T.; Eisenbart, Sara K. Molecular Cell, Vol. 69, Issue 5 https://doi.org/10.1016/j.molcel.2018.01.032	journal	March 2018
Inhibition of CRISPR-Cas9 with Bacteriophage Proteins Rauch, Benjamin J.; Silvis, Melanie R.; Hultquist, Judd F. Cell, Vol. 168, Issue 1-2 https://doi.org/10.1016/j.cell.2016.12.009	journal	January 2017
Naturally Occurring Off-Switches for CRISPR-Cas9 Pawluk, April; Amrani, Nadia; Zhang, Yan Cell, Vol. 167, Issue 7 https://doi.org/10.1016/j.cell.2016.11.017	journal	December 2016
A Broad-Spectrum Inhibitor of CRISPR-Cas9 Harrington, Lucas B.; Doxzen, Kevin W.; Ma, Enbo Cell, Vol. 170, Issue 6 https://doi.org/10.1016/j.cell.2017.07.037	journal	September 2017
Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9 Yang, Hui; Patel, Dinshaw J. Molecular Cell, Vol. 67, Issue 1 https://doi.org/10.1016/j.molcel.2017.05.024	journal	July 2017
A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays Zhang, J. -H. Journal of Biomolecular Screening, Vol. 4, Issue 2 https://doi.org/10.1177/108705719900400206	journal	April 1999
Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System Marshall, Ryan; Maxwell, Colin S.; Collins, Scott P. Molecular Cell, Vol. 69, Issue 1 https://doi.org/10.1016/j.molcel.2017.12.007	journal	January 2018
Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements Karvelis, Tautvydas; Gasiunas, Giedrius; Young, Joshua Genome Biology, Vol. 16, Issue 1 https://doi.org/10.1186/s13059-015-0818-7	journal	November 2015
Deciphering, Communicating, and Engineering the CRISPR PAM Leenay, Ryan T.; Beisel, Chase L. Journal of Molecular Biology, Vol. 429, Issue 2 https://doi.org/10.1016/j.jmb.2016.11.024	journal	January 2017
Small molecules, big targets: drug discovery faces the protein–protein interaction challenge Scott, Duncan E.; Bayly, Andrew R.; Abell, Chris Nature Reviews Drug Discovery, Vol. 15, Issue 8 https://doi.org/10.1038/nrd.2016.29	journal	April 2016

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