RT Journal
A1 Bosch-Guiteras, Núria
A1 Uroda, Tina
A1 Guillen-Ramirez, Hugo A.
A1 Riedo, Rahel
A1 Gazdhar, Amiq
A1 Esposito, Roberta
A1 Pulido-Quetglas, Carlos
A1 Zimmer, Yitzhak
A1 Medová, Michaela
A1 Johnson, Rory
T1 Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs
JF Genome Research 
JO Genome Research 
YR 2021 
FD March 01 
VO 31 
IS 3 
SP 461 
OP 471 
DO 10.1101/gr.265736.120 
UL http://genome.cshlp.org/content/31/3/461.abstract 
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 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.