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Title: Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Abstract

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.

Authors:
ORCiD logo [1];  [1]; ORCiD logo [2];  [3];  [1];  [4]
  1. Univ. of California, Berkeley, CA (United States)
  2. 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)
  3. Gladstone Institutes, San Francisco, CA (United States). Gladstone Institute of Data Science and Biotechnology
  4. 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)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); Defense Advanced Research Projects Agency (DARPA); National Science Foundation (NSF); National Institutes of Health (NIH)
OSTI Identifier:
1633267
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 117; Journal Issue: 12; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
CRISPR; anti-CRISPR; self-targeting; Cas9; genome editing

Citation Formats

Watters, Kyle E., Shivram, Haridha, Fellmann, Christof, Lew, Rachel J., McMahon, Blake, and Doudna, Jennifer A. Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes. United States: N. p., 2020. Web. doi:10.1073/pnas.1917668117.
Watters, Kyle E., Shivram, Haridha, Fellmann, Christof, Lew, Rachel J., McMahon, Blake, & Doudna, Jennifer A. Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes. United States. doi:10.1073/pnas.1917668117.
Watters, Kyle E., Shivram, Haridha, Fellmann, Christof, Lew, Rachel J., McMahon, Blake, and Doudna, Jennifer A. Tue . "Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes". United States. doi:10.1073/pnas.1917668117. https://www.osti.gov/servlets/purl/1633267.
@article{osti_1633267,
title = {Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes},
author = {Watters, Kyle E. and Shivram, Haridha and Fellmann, Christof and Lew, Rachel J. and McMahon, Blake and Doudna, Jennifer A.},
abstractNote = {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.},
doi = {10.1073/pnas.1917668117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
issn = {0027-8424},
number = 12,
volume = 117,
place = {United States},
year = {2020},
month = {3}
}

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