PT  - JOURNAL ARTICLE
AU  - Ryan Clarke
AU  - Robert Heler
AU  - Matthew S. MacDougall
AU  - Nan Cher Yeo
AU  - Alejandro Chavez
AU  - Maureen Regan
AU  - Leslyn Hanakahi
AU  - George M. Church
AU  - Luciano A. Marraffini
AU  - Bradley J. Merrill
TI  - Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks
AID  - 10.1101/300962
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 300962
4099  - http://biorxiv.org/content/early/2018/04/13/300962.short
4100  - http://biorxiv.org/content/early/2018/04/13/300962.full
AB  - 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.