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AcrIIA22 is a novel anti-CRISPR that impairs SpyCas9 activity by relieving DNA torsion of target plasmids

View ORCID ProfileKevin J. Forsberg, View ORCID ProfileDanica T. Schmidtke, Rachel Werther, View ORCID ProfileDeanna Hausman, Barry L. Stoddard, Brett K. Kaiser, View ORCID ProfileHarmit S. Malik
doi: https://doi.org/10.1101/2020.09.28.317578
Kevin J. Forsberg
aDivision of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
bHoward Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
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  • For correspondence: kforsber@fredhutch.org
Danica T. Schmidtke
aDivision of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
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  • ORCID record for Danica T. Schmidtke
Rachel Werther
aDivision of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
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Deanna Hausman
aDivision of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
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  • ORCID record for Deanna Hausman
Barry L. Stoddard
aDivision of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
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Brett K. Kaiser
aDivision of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
cDepartment of Biology, Seattle University, 901 12th Avenue, Seattle, WA 98122, USA
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Harmit S. Malik
aDivision of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
bHoward Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109 USA
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  • ORCID record for Harmit S. Malik
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Abstract

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all mechanistically characterized Acrs directly bind their cognate Cas protein to inactivate CRISPR immunity. Here, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of Clostridial bacteria and their prophages from the human gut microbiome. Uncovered in a functional metagenomic selection, AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic-acid binding proteins. This homology helped us deduce that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids, rendering them less susceptible to SpyCas9, which is highly dependent on negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and mobile genetic elements.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 29, 2020.
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AcrIIA22 is a novel anti-CRISPR that impairs SpyCas9 activity by relieving DNA torsion of target plasmids
Kevin J. Forsberg, Danica T. Schmidtke, Rachel Werther, Deanna Hausman, Barry L. Stoddard, Brett K. Kaiser, Harmit S. Malik
bioRxiv 2020.09.28.317578; doi: https://doi.org/10.1101/2020.09.28.317578
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AcrIIA22 is a novel anti-CRISPR that impairs SpyCas9 activity by relieving DNA torsion of target plasmids
Kevin J. Forsberg, Danica T. Schmidtke, Rachel Werther, Deanna Hausman, Barry L. Stoddard, Brett K. Kaiser, Harmit S. Malik
bioRxiv 2020.09.28.317578; doi: https://doi.org/10.1101/2020.09.28.317578

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