Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.

Bosch-Guiteras, Núria; Uroda, Tina; Guillen-Ramirez, Hugo A.; Riedo, Rahel; Gazdhar, Amiq; Esposito, Roberta; Pulido-Quetglas, Carlos; Zimmer, Yitzhak; Medová, Michaela; Johnson, Rory (2021). Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs. Genome research, 31(3), pp. 461-471. Cold Spring Harbor, N.Y 10.1101/gr.265736.120

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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.

Item Type:

Journal Article (Further Contribution)

Division/Institute:

04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Clinic of Medical Oncology
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR)
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Radio-Onkologie
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Radio-Onkologie

04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Clinic of Radiation Oncology
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Pneumologie (Erwachsene)
04 Faculty of Medicine > Department of Gastro-intestinal, Liver and Lung Disorders (DMLL) > Clinic of Pneumology

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Bosch Guiteras, Nuria; Uroda, Tina; Guillen Ramirez, Hugo Armando; Riedo, Rahel; Gazdhar, Amiq; Esposito, Roberta; Pulido Quetglas, Carlos; Zimmer, Yitzhak; Medova, Michaela and Johnson, Rory Baldwin

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1549-5469

Publisher:

Cold Spring Harbor, N.Y

Language:

English

Submitter:

Heidi Lobsiger

Date Deposited:

17 Feb 2021 10:04

Last Modified:

04 Mar 2021 01:34

Publisher DOI:

10.1101/gr.265736.120

PubMed ID:

33574136

BORIS DOI:

10.48350/152273

URI:

https://boris.unibe.ch/id/eprint/152273

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