Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks

• Published in: Molecular cell 2018-07, Vol.71 (1), p.42-55.e8
• Main Authors: Clarke, Ryan, Heler, Robert, MacDougall, Matthew S., Yeo, Nan Cher, Chavez, Alejandro, Regan, Maureen, Hanakahi, Leslyn, Church, George M., Marraffini, Luciano A., Merrill, Bradley J.
• Format: Article
• Language: English
• Subjects: Animals; Bacteria - genetics; Bacteria - metabolism; Bacteria - virology; Bacteriophages - genetics; Bacteriophages - metabolism; Cas9; Cell Line; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; DNA Breaks, Double-Stranded; DNA Repair; Gene Editing; genome editing; Mice; Mouse Embryonic Stem Cells - metabolism; phage biology; RNA polymerase; strand bias; transcription
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2018.06.005

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-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward 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 protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing. [Display omitted] •Persistent Cas9 binding blocks DNA repair proteins from accessing Cas9-generated breaks•RNA polymerase can dislodge Cas9 from DNA breaks in a highly strand-biased manner•Dislodging Cas9 with RNA polymerase generates multi-turnover nuclease activity•Targeting of Cas9 to phage genome is strand biased toward multi-turnover activities Clarke et al. show that persistent Cas9 binding to double-strand DNA breaks (DSBs) blocks DNA break repair. The Cas9-DSB complex can be disrupted by translocating RNA polymerases in a strand-biased manner, increasing genome editing frequencies and enhancing bacterial immunity to phages through multi-turnover Cas9 cleavage of phage genomes.