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Mol Cell. 2018 Jul 5;71(1):42-55.e8. doi: 10.1016/j.molcel.2018.06.005.

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

Author information

1
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
2
Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.
3
Department of Genetics, Harvard Medical School, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
4
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA; Genome Editing Core, University of Illinois at Chicago, Chicago, IL 60607, USA.
5
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Rockford Health Science Campus, Rockford, IL 61107, USA.
6
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA; Genome Editing Core, University of Illinois at Chicago, Chicago, IL 60607, USA. Electronic address: merrillb@uic.edu.

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-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.

KEYWORDS:

CRISPR; Cas9; DNA repair; RNA polymerase; genome editing; phage biology; strand bias; transcription

PMID:
29979968
PMCID:
PMC6063522
DOI:
10.1016/j.molcel.2018.06.005
[Indexed for MEDLINE]
Free PMC Article

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