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Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks.

Ryan Clarke, Robert Heler, Matthew S MacDougall, Nan Cher Yeo, Alejandro Chavez, Maureen Regan, Leslyn Hanakahi, George M. Church, Luciano A Marraffini, Brad Merrill
doi: https://doi.org/10.1101/300962
Ryan Clarke
University of Illinois at Chicago;
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Robert Heler
The Rockefeller University;
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Matthew S MacDougall
University of Illinois at Chicago;
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Nan Cher Yeo
Harvard Medical School;
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Alejandro Chavez
Harvard Medical School;
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Maureen Regan
University of Illinois at Chicago;
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Leslyn Hanakahi
University of Illinois, Rockford Health Science Campus
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George M. Church
Harvard Medical School;
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Luciano A Marraffini
The Rockefeller University;
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Brad Merrill
University of Illinois at Chicago;
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  • For correspondence: merrillb@uic.edu
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Abstract

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing.

Copyright 
The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
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  • Posted April 13, 2018.

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Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks.
Ryan Clarke, Robert Heler, Matthew S MacDougall, Nan Cher Yeo, Alejandro Chavez, Maureen Regan, Leslyn Hanakahi, George M. Church, Luciano A Marraffini, Brad Merrill
bioRxiv 300962; doi: https://doi.org/10.1101/300962
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Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks.
Ryan Clarke, Robert Heler, Matthew S MacDougall, Nan Cher Yeo, Alejandro Chavez, Maureen Regan, Leslyn Hanakahi, George M. Church, Luciano A Marraffini, Brad Merrill
bioRxiv 300962; doi: https://doi.org/10.1101/300962

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