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-targeting sgRNAs targets a distinct gene, as indicated. For the “LIG4 24hrs” condition, the small fraction of cells that successfully cycled into the subsequent G1 (6.71%) showed approximately half the Hoechst signal (centered at ∼50K) compared with the DAPI signal (centered at ∼100K), consistent with completion of cell division. C. Genome-wide pileup plots for both spontaneous and Cas9-induced on-target SCE events. The targeting site is indicated in the panel with the gene name and a vertical red bar. 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 Figure 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 for the 237-site targeting experiment, comparing the number of SCEs falling within sgRNA-targeted sites (ROI: regions of interest) with randomly sampled sites (“rand”).
“24ws24BrdU” refers to conditions in which cells were treated with Cas9 RNP (or the indicated nickase variants, D10A or H840A) for 24 hours, followed by washout and BrdU labeling for the subsequent 24 hours (or 40 hours, in the case of 24ws40BrdU to allow more cells to progress into the next G1). “24 + 16hrs BrdU” refers to the condition in which Cas9 RNP and BrdU were added simultaneously; after 24 hours of Cas9 RNP treatment, BrdU labeling was continued for an additional 16 hours (a total of 40 hours of BrdU).
RDCP analysis with sgRNA targeting 237 genomic sites.
In sci-L3-Strand-seq plots, reads mapping to the W and C template strands are plotted above and below zero on the y-axis, respectively. SCEs are identified as switches between W and C strand states along a chromosome. 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) shows SV patterns at the SCE junctions consistent with the predicted RDCP signature of SCE mediated by URRs or replication termination zones (green shaded area), although the lagging strands, rather than the leading strands, must be resolved to generate these mitotic breaks.
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 Figure 1. A. Additional FACS plots showing Cas9-induced cell cycle arrest. Local and non-local repair examples. Local fill-in synthesis (1) & NHEJ (2) preserve continuity of the chromatid; SCE requires cis-chromatid disengagement (3) and are coupled with a template strand directionality switch from blue to red or red to blue. B. Additional genome-wide pileup showing on-target SCE. For each sgRNA, top: breakpointR-based SCE calls; boRom: 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 a\er Cas9 RNP washout, corresponding to the second division.
