Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways.

Autor:	Schimmel J; Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands., Muñoz-Subirana N; Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands., Kool H; Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands., van Schendel R; Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands., van der Vlies S; Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands., Kamp JA; Department of Psychiatry, Erasmus Medical Center, Rotterdam, the Netherlands., de Vrij FMS; Department of Psychiatry, Erasmus Medical Center, Rotterdam, the Netherlands., Kushner SA; Department of Psychiatry, Erasmus Medical Center, Rotterdam, the Netherlands; Department of Psychiatry, Columbia University Irving Medical Center, New York, NY, USA., Smith GCM; Artios Pharma, The Glenn Berge Building, Babraham Research Campus, Cambridge, UK., Boulton SJ; Artios Pharma, The Glenn Berge Building, Babraham Research Campus, Cambridge, UK; The Francis Crick Institute, London, UK., Tijsterman M; Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands; Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, the Netherlands. Electronic address: m.tijsterman@lumc.nl.
Jazyk:	angličtina
Zdroj:	Cell reports [Cell Rep] 2023 Feb 28; Vol. 42 (2), pp. 112019. Date of Electronic Publication: 2023 Jan 25.
DOI:	10.1016/j.celrep.2023.112019
Abstrakt:	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. Competing Interests: Declaration of interests S.J.B. is a co-founder, shareholder, and VP of Science Strategy at Artios Pharma, Ltd., Babraham Research Campus, UK. G.C.M.S. is chief scientific officer and shareholder of Artios Pharma, Ltd. (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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