Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic sites in human cells
- Department of Human Genetics, David Geffen School of Medicine, UCLA, United States
Figures
Figure 1 with 1 supplement
Experimental design, cell-cycle perturbation, and genome-wide pileup of both spontaneous and Cas9-induced on-target sister chromatid exchange (SCE) events.
(A) Experimental design. BrdU was incorporated to enable SCE mapping by sci-L3-Strand-seq, either during the same 24 hr window as Cas9 RNP treatment or during a 24 hr period following Cas9 ribonucleoprotein (RNP) washout. Wild-type Cas9 was used unless otherwise indicated for nickase variants. (B) FACS analysis of cell-cycle progression following Cas9 RNP treatment. ‘24 hr’ 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 24 hr‘ condition, the small fraction of cells that successfully cycled into the subsequent G1 (6.71%) showed approximately half the Hoechst signal (centered at ~50 K) compared with the DAPI signal (centered at ~100 K), 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 1—figure supplement 1B. (D) Quantification of on-target SCE events for the four single-site sgRNAs in the first division (24 hr) and in the subsequent division following RNP washout (24 ws).
Figure 1—figure supplement 1
Additional FACS, genome-wide pileup plots.
(A) Additional FACS plots showing Cas9-induced cell-cycle arrest. Local and non-local repair examples. Local fill-in synthesis (1) and non-homologous end joining (NHEJ) (2) preserve continuity of the chromatid; sister chromatid exchange (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; bottom: breakpointR-based and rescued SCE calls. (C) Quantification of spontaneous SCE as a result of polyclonal/bulk Cas9 RNP knockouts (see Supplementary file 1B for pairwise statistical testing).
Figure 2 with 1 supplement
Reciprocal daughter-cell pair (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. Sister chromatid exchanges (SCEs) are identified as switches between W and C strand states along a chromosome. (A) A single reciprocal daughter-cell pair (RDCP) identified from 214 single cells following Cas9 ribonucleoprotein (RNP) treatment targeting 237 sites with concurrent BrdU labeling. (B) Four RDCPs exhibiting large-scale structural variants (SVs) recovered from cells labeled with BrdU after Cas9 RNP washout, corresponding to the second division.
Figure 2—figure supplement 1
Additional reciprocal daughter-cell pair (RDCP) plots with sgRNA targeting 237 genomic sites.
Seven RDCPs without large-scale structural variants (SVs) recovered from cells labeled with BrdU after Cas9 ribonucleoprotein (RNP) washout, corresponding to the second division.
Figure 3 with 1 supplement
Haplotype-aware analysis of observed reciprocal daughter-cell pair (RDCP) (Pair 4, chr1) shows structural variant (SV) patterns at the sister chromatid exchange (SCE) junctions consistent with the predicted RDCP signature of SCE mediated by under-replicated regions (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.
Figure 3—figure supplement 1
Haplotype-aware analysis of reciprocal daughter-cell pair (RDCP) with large-scale structural variants (SVs).
Tables
Table 1
Enrichment analysis for the 237-site targeting experiment.
We compare the number of sister chromatid exchanges (SCEs) falling within sgRNA-targeted sites (ROIs: regions of interest) with randomly sampled sites (‘rand’). ‘24ws24BrdU’ refers to conditions in which cells were treated with Cas9 ribonucleoprotein (RNP) (or the indicated nickase variants, D10A or H840A) for 24 hr, followed by washout and BrdU labeling for the subsequent 24 hr (or 40 hr, in the case of 24ws40BrdU to allow more cells to progress into the next G1). ‘24+16 hr BrdU’ refers to the condition in which Cas9 RNP and BrdU were added simultaneously; after 24 hr of Cas9 RNP treatment, BrdU labeling was continued for an additional 16 hr (a total of 40 hr of BrdU).
| Condition (all cells) | # Cells | On-target ROI | On-target rand | p-Val | Fold ROI/rand | Average # on-target SCE/cell |
|---|---|---|---|---|---|---|
| 237 sites 24ws24BrdU | 74 | 335 | 301 | 0.19 | 1.11 | |
| D10A 237 sites 24ws24BrdU | 330 | 1948 | 1791 | 0.01 | 1.09 | |
| H840A 237 sites 24ws24BrdU | 757 | 3785 | 3707 | 0.37 | 1.02 | |
| 237 sites 24+16 hr BrdU | 214 | 1224 | 1109 | 0.02 | 1.10 | 5.72 |
| 237 sites 24ws40BrdU | 2547 | 15606 | 12994 | 4.89E-55 | 1.20 | 6.13 |
| Condition (# of SCE ≥ 9) | # Cells | On-target ROI | On-target rand | p-Val | Fold ROI/rand | Average # on-target SCE/cell |
| 237 sites 24ws24BrdU | 8 | 62 | 49 | 0.25 | 1.27 | |
| D10A 237 sites 24ws24BrdU | 40 | 447 | 411 | 0.22 | 1.09 | |
| H840A 237 sites 24ws24BrdU | 59 | 440 | 401 | 0.18 | 1.10 | |
| 237 sites 24+16 hr BrdU | 4 | 46 | 33 | 0.17 | 1.39 | 11.50 |
| 237 sites 24ws40BrdU | 261 | 3812 | 2746 | 1.29E-41 | 1.39 | 14.61 |
Additional files
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Supplementary file 1
File 1A.
Comparisons of sister chromatid exchange (SCE) levels among single-gene editing conditions. File 1B. Description of structural variant (SV) for reciprocal daughter-cell pairs (RDCPs) shown in Figure 2B. DC: daughter cell. File 1C. Summary of all 15 RDCPs.
- https://cdn.elifesciences.org/articles/112073/elife-112073-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/112073/elife-112073-mdarchecklist1-v1.pdf
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Source code 1
Scripts to reproduce analyses presented in the manuscript.
- https://cdn.elifesciences.org/articles/112073/elife-112073-code1-v1.zip
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Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic sites in human cells
eLife 15:RP112073.
https://doi.org/10.7554/eLife.112073.3
