Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

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 falsenegative 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.

Deletion mapping of regulatory elements for GATA3 reveals a distal T helper 2 cell enhancer involved in allergic diseases

“…We note however, that due to variation in guide efficiency and non-homologous end joining (NHEJ), paired guides introduce spanning deletions only some fraction of the time. Instead, they often introduce small insertions/deletions (indels) at each of the target sites 32,33 . To address this, we estimated the deletion efficiency of our system by performing paired-guide mediated deletions of several 1-2 kb sequences in the 2 Mb survey region and measuring the deletion efficiency by quantitative PCR (qPCR) (Supplementary Fig.…”

Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells

“…To explain this observation, we considered the presence of (1) heterozygous deletion clones (Canver et al 2014), whereby one allele carries the deletion and the other allele is unmodified, (2) mutations occurring in the PAM sequences (Mali et al 2013;Sternberg et al 2014) preventing the recruitment of Cas9 and its cutting activity at the gene locus, and (3) unsynchronized Cas9 cleavage at the two DSB sites (Bosch-Guiteras et al 2021), in which one DSB would have been repaired before the induction of the second DSB. These three scenarios can be excluded because we would have detected a PCR product corresponding to the size of the unmodified allele (Supplemental Fig.…”

CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells

“…To explain this observation, we considered several possibilities that can lead to imperfect genomic Cas9 deletions in cells. Previous reports described that applications of the dual gRNA system (I) could lead to heterozygous deletion clones (8) whereby one allele carries the deletion and the other allele is unmodified, (II) mutations could have occurred at the PAM sequences (44, 45) preventing the recruitment of Cas9 and its cutting activity at the gene locus, (III) the Cas9 cleavage might be unsynchronized at the two DSB sites (46), in which one DSB would have been repaired before the induction of the second DSB or the target sequence could have been excised and then either (IV) inverted or (V) duplicated (8, 13, 47–49). Scenarios I to IV can be excluded since we would have detected a PCR product corresponding to the size of the unmodified allele (ca.…”

“…Previous reports described genomic aberrations when using the Cas9 dual gRNA system. For example, (I) the application could lead to heterozygous deletion clones (8) whereby one allele carries the deletion and the other allele is unmodified, (II) mutations could occur at the PAM sequences (54,55) preventing the recruitment of Cas9 and its cutting activity at the gene locus, (III) the Cas9 cleavage might be unsynchronized at the two DSB sites (56), in which one DSB would have been repaired before the induction of the second DSB or the target sequence could have been excised and then either (IV) inverted or (V) duplicated (8,13,(57)(58)(59). However, to our knowledge a combination of a simultaneously occurring inversion and duplication has not been described.…”