Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic targets

Reviewer #1 (Public review):

Summary:

This manuscript uses sci-L3-Strand-seq to map sister chromatid exchange events following CRISPR/Cas9-induced DNA damage. Because exchanges between identical sister chromatids are largely invisible to conventional sequencing, the study addresses an important blind spot in the assessment of genome editing outcomes. The authors compare single-locus Cas9 cleavage, simultaneous targeting of 237 repetitive genomic sites, and Cas9 nickase variants. They further use reciprocal daughter-cell pair analysis to ask whether Cas9-associated SCEs are copy-neutral or linked to larger structural alterations. Overall, this is a valuable study that introduces an important additional layer to the analysis of CRISPR/Cas9 repair outcomes. The central finding that Cas9-induced DSBs can trigger frequent local SCE is well supported and likely to be of broad interest. The evidence for structural complexity associated with some induced SCEs is intriguing, but the mechanistic interpretation should either be tested directly or presented more cautiously.

Strengths:

The major strength of the manuscript is the application of a strand-resolved, single-cell method to a question that is difficult to address with standard genome sequencing. The evidence that a single Cas9-induced DSB can trigger strong local SCE is compelling in concept and supported by multiple guide RNAs targeting distinct loci. The reported on-target SCE frequencies, reaching up to 41%, suggest that inter-sister exchange is a substantial and underappreciated outcome of Cas9 cleavage.

Of particular interest is the comparison between single-site and multi-site targeting. The finding that 237 programmed Cas9 targets produce only mild bulk enrichment of on-target SCE but stronger enrichment in a subpopulation of cells with elevated SCE burden is interesting and may have wider biological implications, particularly if the findings extend beyond Cas9-induced SCE to spontaneous SCEs. Given that potential, the current manuscript would benefit greatly from any experiments characterizing this sub-population: are these cells in a particular cell cycle state, experiencing changes in gene expression, or do they have other unique biological properties?

The reciprocal daughter-cell pair analysis is another notable feature of the study. The observation that some Cas9-associated SCEs are accompanied by structural alterations could challenge the assumption that SCE after a programmed break reflects error-free homologous recombination.

Weaknesses:

The number of informative RDCPs is limited, and the mechanistic interpretation of the "WWC-or-WCC/deletion" signature is more suggestive than definitive. In particular, the manuscript invokes (even though only in the Discussion section) URR or replication-termination-zone resolution and discusses TRAIP-dependent CMG unloading, nuclease cleavage, and polymerase theta-mediated joining, but these pathway components are not directly tested herein. A more conservative conclusion that some Cas9-associated SCEs coincide with structural alterations is more appropriate, particularly in the Discussion and Conclusion. For example, the statement that this work provides "direct genetic evidence" for a URR-type mechanism is overstated unless supported by additional experiments or a more extensive analysis of alternative models. Similarly, while the authors explain the limitations of acute Cas9 disruption of LIG3, LIG4, XRCC1, and XRCC4, the manuscript should clarify what biological questions this experiment can and cannot answer.

Reviewer #2 (Public review):

Summary:

In this short paper, a clever single-cell Strand-seq method was used to study the number and location of sister chromatid exchange events (SCEs) in cells after CRISPR/Cas9-induced DNA double-strand breaks (DSBs). Unique as well as multiple genomic loci were targeted. Cas9-induced cuts at unique genomic locations led to statistical enrichment of SCEs at the target site, whereas Cas9 targeted at repetitive targets revealed only mild enrichment of on-target SCEs unless analysis was restricted to a subset of cells with >8 SCEs per cell. Interestingly, reciprocal daughter-cell pair analysis revealed large-scale structural alterations on some chromosomes. Whereas disruption of DNA repair genes, including LIG3, LIG4, XRCC1, and XRCC4, did not measurably alter SCE frequency per cell within 24 hrs, consistent with delayed functional loss following editing and selection against essential genes. Together, these findings demonstrate that Cas9-induced DSBs are potent local triggers of SCE at unique loci and can be associated with structural alterations, highlighting the influence of lesion type and genomic context on recombination outcomes during genome editing.

Strengths:

The data in this paper represent a very rich resource of how parental DNA template strands are distributed in paired daughter cells after various treatments. Abnormalities observed in only one of such paired daughter cells provide a novel and exciting approach to study mechanisms of DNA instability and DNA repair at a genome-wide level in general and following Cas9-induced DSB in particular.

Weaknesses:

The effect of Cas9-induced DSBs in the cells that are used will depend on the cell cycle stage of the cells that are targeted, as well as the number of times cuts are made. The latter could happen before, during, and after DNA repair reactions on one or both alleles in a diploid cell. As a result, it is very difficult to extrapolate the mechanisms of DNA instability and DNA repair from the observed genomic rearrangements. Novel approaches are needed to limit the number and timing of Cas9-induced breaks to overcome some of these limitations. The language and logic in the paper can be improved, and some of the claims seem incorrect. For example, the abstract reads "A single Cas9 cut at a unique genomic locus led to strong local enrichment of SCE at the break site, reaching up to 41% in the same cell cycle and 17% in the subsequent division, indicating that DSB repair frequently engages non-local inter-sister repair." The evidence that only a single Cas9 cut was made is lacking (see my earlier comment); it is not clear how local enrichment or non-local inter-sister repair are defined.

Reviewer #3 (Public review):

Summary:

Chovanec and Yin used their newly developed sci-L3-Strand-seq powerful method to characterize SCE after Cas9 cleavage in a human cell line, using either a single target site or an element repeated 237 times in the genome. SCE are often neglected in DNA repair analyses since they are “genetically silent”. Interestingly, the authors found enrichment of SCE at unique Cas9 sites, but only a modest enrichment of SCE when Cas9 targets 237 sites in the genome. The genetic control of SCE formation at Cas9 sites is not deliberately addressed in this paper. However, the authors found that targeted SCE seem to be enriched in a subpopulation of cells, particularly “permissive” for SCE, but the determinants of such a population are unknown. Finally, the power of the sci-L3-Strand-seq allowed the authors to characterize a specific type of SCE based on the analysis of reciprocal daughter-cell pairs' genomes that is associated with a specific type of chromosomal rearrangement compatible with the ones observed in HR defective BRCA1/2 deficient cells.

Strengths:

This is an interesting paper that molecularly explores sister chromatid exchanges, which represent an important challenge in molecular biology since they are genetically silent.

Weaknesses:

A complexity of the current paper is that it heavily relies on a recently published paper (Chovanec et al 2026, NAR) describing the powerful but complex technique sci-L3-Strand-seq. Knowledge of this paper is a prerequisite to understanding the current manuscript because no reminder is provided. In addition, the current manuscript presents the use of the sci-L3-Strand-seq technique in the study of SCE after Cas9-induced DSBs, while a companion study is referred to several times for containing results about SCE in XRCC1 KO. At some point, one questions the relevance of splitting the use of sci-L3-Strand-seq in different papers instead of making a single integrated one.

Author response:

We thank the editors and reviewers for their thoughtful evaluation and constructive feedback. We are pleased that all three reviewers recognize the importance of mapping Cas9-induced sister chromatid exchanges (SCE) as a previously invisible repair outcome, and that the RDCP analysis is a notable feature of the study.

We note that since our manuscript was posted, two companion studies in Science have provided direct biochemical evidence for the TRAIP-dependent pathway we discussed:

(1) Fujisawa & Labib (Science, 2026; DOI: 10.1126/science.aeh2300) showed that TTF2 bridges CDK1-phosphorylated TRAIP to DNA Polymerase epsilon in the replisome, triggering mitotic CMG helicase disassembly, fork cleavage, and repair via SCE. Loss of this pathway reduced replication stress-induced SCE approximately two-fold in mouse ES cells.

(2) Can et al. (Science, 2026; DOI: 10.1126/science.aeh1834) independently identified the same CDK1-TTF2-TRAIP axis in Xenopus egg extracts and validated it in HCT116 cells, showing that disrupting the TRAIP-TTF2 interaction reduced common fragile site deletions.

We will incorporate these references in the revised discussion while still framing our RDCP observations as consistent with, rather than definitive proof of, this pathway.

Below we briefly address the main points raised in the public reviews.

Reviewer #1:

We agree that the mechanistic interpretation of the RDCP signature should be presented more cautiously. We will reframe the URR/TRAIP discussion as a model, replacing language such as "direct genetic evidence" with "consistent with." We will add a summary table of RDCP data. We will also expand the description of rescued SCE calls. We will clarify what the DNA repair gene targeting experiment can and cannot answer (delayed protein loss, essential-gene selection) - we think that there is a notable difference at the bulk vs. at the single-cell level depending on the nature of the assay. Fig.1 fonts, labels, and pileup plot descriptions will be improved.

We agree that the high-SCE subpopulation is particularly interesting and we cannot currently distinguish higher RNP uptake, a permissive cell-cycle state, altered expression, or stochastic variation. This may be better explored by future co-assays with sci-L3-Strand-seq.

Reviewer #2:

We agree with the limitations that Cas9-induced DSBs can be dependent on the cell cycle stage and the number of times cuts are made. We will add a brief discussion on this limitation in extrapolating the mechanisms of DNA instability and DNA repair from the observed genomic rearrangements.

We agree that "a single Cas9 cut" should be revised to "Cas9 targeting of a single genomic locus" to accurately reflect the experimental design. We will clarify the possibility of multiple rounds of cutting at the same sites. We will also clarify "non-local" by modifying Fig.1 - we used this term specifically to include the possibility of inter-sister NHEJ.

Reviewer #3:

We will improve the self-contained nature of the manuscript so that readers need not consult the earlier NAR papers, and the companion preprint to understand the key results.