TY - JOUR T1 - Enhancing CRISPR deletion via pharmacological delay of DNA-PK JF - bioRxiv DO - 10.1101/2020.02.12.945907 SP - 2020.02.12.945907 AU - Núria Bosch AU - Michaela Medová AU - Roberta Esposito AU - Carlos Pulido-Quetglas AU - Yitzhak Zimmer AU - Rory Johnson Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/02/13/2020.02.12.945907.abstract N2 - 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. ER -