PT  - JOURNAL ARTICLE
AU  - Núria Bosch
AU  - Michaela Medová
AU  - Roberta Esposito
AU  - Carlos Pulido-Quetglas
AU  - Yitzhak Zimmer
AU  - Rory Johnson
TI  - Enhancing CRISPR deletion via pharmacological delay of DNA-PK
AID  - 10.1101/2020.02.12.945907
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.02.12.945907
4099  - http://biorxiv.org/content/early/2020/05/27/2020.02.12.945907.short
4100  - http://biorxiv.org/content/early/2020/05/27/2020.02.12.945907.full
AB  - 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 non-homologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false negative results. Using an endogenous reporter system, we demonstrate that temporary inhibition of DNA-dependent protein kinase (DNA-PK) – an early step in NHEJ - yields up to 17-fold increase 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. Importantly, the method is compatible with pooled functional screens employing lentivirally-delivered guide RNAs. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.Competing Interest StatementThe authors have declared no competing interest.