Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Watters, Kyle E.; Shivram, Haridha; Fellmann, Christof; Lew, Rachel J.; McMahon, Blake; Doudna, Jennifer A.

doi:10.1073/pnas.1917668117

Title: Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Journal Article · Tue Mar 10 00:00:00 EDT 2020 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1917668117· OSTI ID:1633267

^[1]; Shivram, Haridha ^[1];

^[2]; Lew, Rachel J. ^[3]; McMahon, Blake ^[1]; Doudna, Jennifer A. ^[4]

Univ. of California, Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States); Gladstone Institutes, San Francisco, CA (United States). Gladstone Institute of Data Science and Biotechnology; Univ. of California, San Francisco, CA (United States)
Gladstone Institutes, San Francisco, CA (United States). Gladstone Institute of Data Science and Biotechnology
Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Institute and Innovative Genomics Institute; Gladstone Institutes, San Francisco, CA (United States). Gladstone Institute of Data Science and Biotechnology; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13–AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13–AcrIIA15 as unique bifunctional inhibitors of SauCas9.

View Accepted Manuscript (DOE)

Cite

Export

Save

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

Sponsoring Organization:: USDOE Office of Science (SC); Defense Advanced Research Projects Agency (DARPA); National Science Foundation (NSF); National Institutes of Health (NIH)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1633267

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, Issue 12; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 36 works

Citation information provided by
Web of Science

References (43)

An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9 Hynes, Alexander P.; Rousseau, Geneviève M.; Lemay, Marie-Laurence Nature Microbiology, Vol. 2, Issue 10 https://doi.org/10.1038/s41564-017-0004-7	journal	August 2017
Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system Bondy-Denomy, Joe; Pawluk, April; Maxwell, Karen L. Nature, Vol. 493, Issue 7432 https://doi.org/10.1038/nature11723	journal	December 2012
Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing Garcia, Bianca; Lee, Jooyoung; Edraki, Alireza Cell Reports, Vol. 29, Issue 7 https://doi.org/10.1016/j.celrep.2019.10.017	journal	November 2019
CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification Oakes, Benjamin L.; Fellmann, Christof; Rishi, Harneet Cell, Vol. 176, Issue 1-2 https://doi.org/10.1016/j.cell.2018.11.052	journal	January 2019
Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo Park, Hyo Min; Liu, Hui; Wu, Joann Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-05641-3	journal	August 2018
Study the Features of 57 Confirmed CRISPR Loci in 38 Strains of Staphylococcus aureus Zhao, Xihong; Yu, Zhixue; Xu, Zhenbo Frontiers in Microbiology, Vol. 9 https://doi.org/10.3389/fmicb.2018.01591	journal	July 2018
Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a Knott, Gavin J.; Cress, Brady F.; Liu, Jun-Jie eLife, Vol. 8 https://doi.org/10.7554/eLife.49110	journal	August 2019
Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems Hwang, Sungwon; Maxwell, Karen L. The CRISPR Journal, Vol. 2, Issue 1 https://doi.org/10.1089/crispr.2018.0052	journal	February 2019
A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa Pawluk, April; Bondy-Denomy, Joseph; Cheung, Vivian H. W. mBio, Vol. 5, Issue 2 https://doi.org/10.1128/mBio.00896-14	journal	April 2014
Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species Pawluk, April; Staals, Raymond H. J.; Taylor, Corinda Nature Microbiology, Vol. 1, Issue 8 https://doi.org/10.1038/nmicrobiol.2016.85	journal	June 2016
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
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
A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator Athukoralage, Januka S.; Graham, Shirley; Grüschow, Sabine Journal of Molecular Biology, Vol. 431, Issue 15 https://doi.org/10.1016/j.jmb.2019.04.041	journal	July 2019
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
Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity He, Fei; Bhoobalan-Chitty, Yuvaraj; Van, Lan B. Nature Microbiology, Vol. 3, Issue 4 https://doi.org/10.1038/s41564-018-0120-z	journal	March 2018
A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core Zimmermann, Lukas; Stephens, Andrew; Nam, Seung-Zin Journal of Molecular Biology, Vol. 430, Issue 15 https://doi.org/10.1016/j.jmb.2017.12.007	journal	July 2018
Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems: Enhancing TXTL-Based Expression With χ-Site DNA Marshall, Ryan; Maxwell, Colin S.; Collins, Scott P. Biotechnology and Bioengineering, Vol. 114, Issue 9 https://doi.org/10.1002/bit.26333	journal	May 2017
Inhibition of Type III CRISPR-Cas Immunity by an Archaeal Virus-Encoded Anti-CRISPR Protein Bhoobalan-Chitty, Yuvaraj; Johansen, Thomas Baek; Di Cianni, Nadia Cell, Vol. 179, Issue 2 https://doi.org/10.1016/j.cell.2019.09.003	journal	October 2019
Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins Zhu, Yalan; Gao, Ang; Zhan, Qi Molecular Cell, Vol. 74, Issue 2 https://doi.org/10.1016/j.molcel.2019.01.038	journal	April 2019
Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent Proteins ^† Ai, Hui-wang; Shaner, Nathan C.; Cheng, Zihao Biochemistry, Vol. 46, Issue 20 https://doi.org/10.1021/bi700199g	journal	May 2007
Genome scale screening identification of SaCas9/gRNAs for targeting HIV-1 provirus and suppression of HIV-1 infection Wang, Qiankun; Liu, Shuai; Liu, Zhepeng Virus Research, Vol. 250 https://doi.org/10.1016/j.virusres.2018.04.002	journal	May 2018
Discovery of widespread type I and type V CRISPR-Cas inhibitors Marino, Nicole D.; Zhang, Jenny Y.; Borges, Adair L. Science, Vol. 362, Issue 6411 https://doi.org/10.1126/science.aau5174	journal	September 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
PHASTER: a better, faster version of the PHAST phage search tool Arndt, David; Grant, Jason R.; Marcu, Ana Nucleic Acids Research, Vol. 44, Issue W1 https://doi.org/10.1093/nar/gkw387	journal	May 2016
An anti-CRISPR protein disables type V Cas12a by acetylation Dong, Liyong; Guan, Xiaoyu; Li, Ningning Nature Structural & Molecular Biology, Vol. 26, Issue 4 https://doi.org/10.1038/s41594-019-0206-1	journal	April 2019
Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci Chen, Baohui; Hu, Jeffrey; Almeida, Ricardo Nucleic Acids Research, Vol. 44, Issue 8 https://doi.org/10.1093/nar/gkv1533	journal	January 2016
Islander: a database of precisely mapped genomic islands in tRNA and tmRNA genes Hudson, Corey M.; Lau, Britney Y.; Williams, Kelly P. Nucleic Acids Research, Vol. 43, Issue D1 https://doi.org/10.1093/nar/gku1072	journal	November 2014
Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome Forsberg, Kevin J.; Bhatt, Ishan V.; Schmidtke, Danica T. eLife, Vol. 8 https://doi.org/10.7554/eLife.46540	journal	September 2019
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
An Optimized microRNA Backbone for Effective Single-Copy RNAi Fellmann, Christof; Hoffmann, Thomas; Sridhar, Vaishali Cell Reports, Vol. 5, Issue 6 https://doi.org/10.1016/j.celrep.2013.11.020	journal	December 2013
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
Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription Stanley, Sabrina Y.; Borges, Adair L.; Chen, Kuei-Ho Cell, Vol. 178, Issue 6 https://doi.org/10.1016/j.cell.2019.07.046	journal	September 2019
Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins Hynes, Alexander P.; Rousseau, Geneviève M.; Agudelo, Daniel Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-05092-w	journal	July 2018
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
Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla Uribe, Ruben V.; van der Helm, Eric; Misiakou, Maria-Anna Cell Host & Microbe, Vol. 25, Issue 2 https://doi.org/10.1016/j.chom.2019.01.003	journal	February 2019
CRISPR-Cas guides the future of genetic engineering Knott, Gavin J.; Doudna, Jennifer A. Science, Vol. 361, Issue 6405 https://doi.org/10.1126/science.aat5011	journal	August 2018
Broad-spectrum enzymatic inhibition of CRISPR-Cas12a Knott, Gavin J.; Thornton, Brittney W.; Lobba, Marco J. Nature Structural & Molecular Biology, Vol. 26, Issue 4 https://doi.org/10.1038/s41594-019-0208-z	journal	April 2019
A Unified Resource for Tracking Anti-CRISPR Names Bondy-Denomy, Joseph; Davidson, Alan R.; Doudna, Jennifer A. The CRISPR Journal, Vol. 1, Issue 5 https://doi.org/10.1089/crispr.2018.0043	journal	October 2018
Anti‐CRISPRs: The natural inhibitors for CRISPR‐Cas systems Zhang, Fei; Song, Guoxu; Tian, Yong Animal Models and Experimental Medicine https://doi.org/10.1002/ame2.12069	journal	May 2019
The Interfaces of Genetic Conflict Are Hot Spots for Innovation Carter, Joshua; Hoffman, Connor; Wiedenheft, Blake Cell, Vol. 168, Issue 1-2 https://doi.org/10.1016/j.cell.2016.12.007	journal	January 2017
Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla Uribe, Ruben V.; van der Helm, Eric; Misiakou, Maria-Anna Cell Host & Microbe, Vol. 26, Issue 5 https://doi.org/10.1016/j.chom.2019.09.005	journal	November 2019
Cheese, phages and anti-CRISPRs Davidson, Alan R. Nature Microbiology, Vol. 2, Issue 10 https://doi.org/10.1038/s41564-017-0026-1	journal	September 2017
Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins Lee, Jooyoung; Mir, Aamir; Edraki, Alireza mBio, Vol. 9, Issue 6 https://doi.org/10.1128/mbio.02321-18	journal	December 2018

Cited By (2)

Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14 Gabel, Clinton; Li, Zhuang; Zhang, Heng Nucleic Acids Research, Vol. 49, Issue 1 https://doi.org/10.1093/nar/gkaa1199	journal	December 2020
Machine learning predicts new anti-CRISPR proteins Eitzinger, Simon; Asif, Amina; Watters, Kyle E. Nucleic Acids Research, Vol. 48, Issue 9 https://doi.org/10.1093/nar/gkaa219	journal	April 2020