2. Academic Validation
  3. Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

  • Genome Res. 2021 Mar;31(3):461-471. doi: 10.1101/gr.265736.120.
Núria Bosch-Guiteras 1 2 3,  Tina Uroda  # 1 2,  Hugo A Guillen-Ramirez  # 1 2,  Rahel Riedo 2 4,  Amiq Gazdhar 2 5,  Roberta Esposito 1 2,  Carlos Pulido-Quetglas 1 2 3,  Yitzhak Zimmer 2 4,  Michaela Medová 2 4,  Rory Johnson 1 2 6 7
Affiliations

Affiliations

  • 1 Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.
  • 2 Department for BioMedical Research, University of Bern, 3008 Bern, Switzerland.
  • 3 Graduate School of Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland.
  • 4 Department of Radiation Oncology, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.
  • 5 Department of Pulmonary Medicine, University Hospital Bern, University of Bern, 3008 Bern, Switzerland.
  • 6 School of Biology and Environmental Science, University College Dublin, Dublin D04 V1W8, Ireland.
  • 7 Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin D04 V1W8, Ireland.
  • # Contributed equally.
PMID: 33574136 DOI: 10.1101/gr.265736.120
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.

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