Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs
- Núria Bosch-Guiteras1,2,3,
- Tina Uroda1,2,8,
- Hugo A. Guillen-Ramirez1,2,8,
- Rahel Riedo2,4,
- Amiq Gazdhar2,5,
- Roberta Esposito1,2,
- Carlos Pulido-Quetglas1,2,3,
- Yitzhak Zimmer2,4,
- Michaela Medová2,4 and
- Rory Johnson1,2,6,7
- 1Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland;
- 2Department for BioMedical Research, University of Bern, 3008 Bern, Switzerland;
- 3Graduate School of Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland;
- 4Department of Radiation Oncology, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland;
- 5Department of Pulmonary Medicine, University Hospital Bern, University of Bern, 3008 Bern, Switzerland;
- 6School of Biology and Environmental Science, University College Dublin, Dublin D04 V1W8, Ireland;
- 7Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin D04 V1W8, Ireland
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↵8 These authors contributed equally to this work.
Abstract
CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)—an early step in NHEJ—yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.265736.120.
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Freely available online through the Genome Research Open Access option.
- Received May 7, 2020.
- Accepted January 15, 2021.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
