Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks

Abstract

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

Poly(ADP) ribose polymerase promotes DNA polymerase theta-mediated end joining by activation of end resection

“…2a). In accord with previous work 9,10,28 , R26 MHD is reduced 4-fold in Polq-deficient contexts (either Polq-/-cells, or in cells pre-treated with Polθi) (Fig. 2b), relative to wild-type controls.…”

“…In sum, significant effects of PARP inhibition appear confined to an impairment of short-range resection and repair by TMEJ, and apparent re-channeling to another repair fate. Consistent with this argument, an NHEJ-specific signature repair product at this locus (an insertion of a single nucleotide; reduced 50-fold in Ku-deficient cells 28 ) was enriched in cells treated with olaparib (Fig. 5d, Supplementary Fig.…”

“…However, it is not possible to employ signature products to accurately estimate whether compensation is complete; thus, we sequenced all repair involving insertions and deletions (indels). We classified the TMEJ fraction as all deletions significantly reduced in Polq−/− cells 10 and the NHEJ fraction as all indels less than 5 bp without microhomology 2 bp or more 28 (Fig. 6a).…”

“…Variant clones of the WT MEFs deficient in Parp1 (Parp1−/−) were generated using Cas9 and the guide listed in Supplementary Table 1; Parp2 deficiency was generated in a Parp1−/− clonal line by a subsequent introduction of Cas9 and the guide listed in Supplementary Table 1 to generate a clonal line deficient in Parp1 and Parp2 (Parp1/2−/−). Mre11 ATLD1 MEFs were generated by bi-allelic knock-in and previously characterized 28 . Retinal pigment epithelial (RPE-1) cells immortalized by human telomerase reverse transcriptase (hTERT) expression were cultured in DMEM F12 (Invitrogen) containing 10% Fetal Bovine Serum (VWR Life Sciences, Seradigm) and penicillin (5 U/mL, Sigma).…”

SIQ: easy quantitative measurement of mutation profiles in sequencing data

“…DNA from 6-TG-resistant cells was isolated for each cell line and amplified using specific primers ( Supplementary Table S1 ). Deep sequencing was performed on these amplicons and the data were analysed by SIQ, and in parallel by CRISPResso2, AmpliCan and ScarMapper ( 19 ) to compare the performances on real data ( Supplementary Figure S2 ). Importantly, the cellular selection we applied (i.e.…”