Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways
Joost Schimmel,
Núria Muñoz-Subirana,
Hanneke Kool,
Robin van Schendel,
Sven van der Vlies,
Juliette A. Kamp,
Femke de Vrij,
Steven A. Kushner,
Graeme C.M. Smith,
Simon J. Boulton,
Marcel Tijsterman
Affiliations
Joost Schimmel
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
Núria Muñoz-Subirana
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
Hanneke Kool
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
Robin van Schendel
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
Sven van der Vlies
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
Juliette A. Kamp
Department of Psychiatry, Erasmus Medical Center, Rotterdam, the Netherlands
Femke de Vrij
Department of Psychiatry, Erasmus Medical Center, Rotterdam, the Netherlands
Steven A. Kushner
Department of Psychiatry, Erasmus Medical Center, Rotterdam, the Netherlands; Department of Psychiatry, Columbia University Irving Medical Center, New York, NY, USA
Graeme C.M. Smith
Artios Pharma, The Glenn Berge Building, Babraham Research Campus, Cambridge, UK
Simon J. Boulton
Artios Pharma, The Glenn Berge Building, Babraham Research Campus, Cambridge, UK; The Francis Crick Institute, London, UK
Marcel Tijsterman
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands; Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, the Netherlands; Corresponding author
Summary: Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.
