Figures and data
Experimental design, cell cycle perturbation and genome-wide pileup of both spontaneous and Cas9-induced on-target SCE events.
A. Experimental design. BrdU was incorporated to enable SCE mapping by sci-L3-Strand-seq, either during the same 24-hour window as Cas9 RNP treatment or during a 24-hour period following Cas9 RNP washout. Wild-type Cas9 was used unless otherwise indicated for nickase variants. B. FACS analysis of cell-cycle progression following Cas9 RNP treatment. “24hrs” denotes concurrent Cas9 RNP treatment and BrdU labeling, whereas “24 ws BrdU” indicates BrdU labeling after Cas9 RNP washout. Each of the four single-cut sgRNAs targets a distinct gene, as indicated. C. Genome-wide pileup plots for both spontaneous and Cas9-induced on-target SCE events. For sgRNAs targeting XRCC1 and XRCC4, SCEs from both the first division during RNP treatment and the post-washout division are shown. For LIG3 and LIG4, SCEs from the first division are shown. Additional pileups, including comparisons between breakpointR-based and rescued SCE calls, are provided in Fig.S1B. D. Quantification of on-target SCE events for the four single-site sgRNAs in the first division (24 hrs) and in the subsequent division following RNP washout (24 ws).
Enrichment analysis of the number of SCE falling within sgRNA targeted sites, i.e., ROI: regions of interest vs. randomly sampled sites (“rand”).
RDCP analysis with sgRNA targeting 237 genomic sites.
A. A single RDCP identified from 214 single cells following Cas9 RNP treatment targeting 237 sites with concurrent BrdU labeling. B. Four RDCPs exhibiting large-scale SVs recovered from cells labeled with BrdU after Cas9 RNP washout, corresponding to the second division.
Haplotype-aware analysis of observed RDCP (Pair 4, chr1) provides direct genetic evidence of predicted RDCP signature of SCE mediated by URR or replication termination zone (green shaded area), although the cut has to happen on the lagging strand.
Oval depicts centromere. D1: daughter cell 1. D1 has a deletion region at the SCE junction. The corresponding region in D2 has a WC region within the haplotype with the SCE (WWC if depicted together with the other haplotype) as both strands are segregated without replication. Details in text.
Related to Fig. 1.
A. Additional FACS plots showing Cas9-induced cell cycle arrest. B. Additional genome-wide pileup showing on-target SCE. For each sgRNA, top: breakpointR-based SCE calls; bottom: breakpointR-based and rescued SCE calls. C. Quantification of spontaneous SCE as a result of polyclonal/bulk Cas9 RNP knockouts (see Tab.S2 for pair-wise statistical testing).
Additional RDCP plots with sgRNA targeting 237 genomic sites.
Seven RDCPs without large-scale SVs recovered from cells labeled with BrdU after Cas9 RNP washout, corresponding to the second division.
