A CRISPR-Cas9-based system for the dose-dependent study of DNA double-strand breaks sensing and repair

  1. Jocelyn Coiffard
  2. Sylvain Kumanski
  3. Olivier Santt
  4. Benjamin Pardo
  5. María Moriel-Carretero

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Abstract

The integrity of DNA is put at risk by different lesions, among which double strand breaks (DSBs) occur at low frequency, yet remain one of the most life-threatening harms. The study of DSB repair requests tools provoking their accumulation, and include the use of chemical genotoxins, ionizing radiations or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nuclease expression permits assessments at precise locations. In this work, we have exploited the repetitiveness of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled induction of 0, 1, 15 or 59 DSBs in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1 in the nuclear space. We found that Tel1 is capable of forming nuclear foci, which are clustered by condensing when DSBs occur in Ty elements. In striking contrast with other DSB-related protein foci, Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    DNA double-strand breaks are harmful to cells. The authors used CRISPR-Cas9 to create DSBs in repetitive elements (Ty transposons) in the Saccharomyces cerevisiae genome. This method builds on previous ones that used the HO endonuclease or prokaryotic restriction enzymes. They used Cas9-based DSBs to assess the role of Tel1 (ATR) in DSB sensing, comparing it with the DSB-generating xenobiotic zeocin. The first part of the paper is fine, but it lacks information about the yield. The system is not as efficient as it is claimed to be, and it is slow. The second part of the paper is not well connected to the first half, and several controls are needed to make it sound.

    Major comments:

    1. A significant portion of the manuscript posits that the CRISPR-Cas9 system is capable of creating as many DSBs as the theoretical number of Ty targets. Nevertheless, it is clear that this is not the case. The authors can determine the number of breaks per construction. A plot of the frequency of average DSBs in chromosomes IV and III appears to be feasible based on the Southern blots presented, although this could give an underestimate, given that some broken chromosomes may become trapped in the well during DSB processing. However, the percentage of zero DSBs for a given chromosome is readily quantifiable, allowing the frequency of DSBs per cell to be determined through straightforward calculations. Once this has been achieved, the authors should revisit their assessments of Rfa1/Rad52/Tel1 foci formation and number.

    2. OPTIONAL: It seems plausible that a saturation of DSBs per cell may occur when the actual number of DSBs is plotted against the theoretical maximum. In such a scenario, the introduction of additional Cas9 molecules could enhance the efficiency of DSB generation. This could be achieved by increasing the number of copies of the CAS9 gene.

    3. The majority of the discussion chapter is dedicated to the second part of the paper (Tel1), yet no mention is made of the subject I have just commented on. Similarly, a comparison should be made between the Cas9-based system and previous methodologies for creating single and multiple DSBs (HO, I-SceI, restriction enzymes, radiation, radiomimetic drugs, etc.) in terms of efficiency, time to DSB, cell response, etc. It is surprising that the Cas9 system takes so long to generate DSBs and that the cell cycle profile indicates very little arrest in G2/M. This should also be discussed.

    4. OPTIONAL: Since the lack of a strong G2/M arrest is intriguing, it would be good to do a Western blot of Rad53 to learn more about Cas9-based DSB sensing and the DNA damage checkpoint. Comparing it to both the well-established HO system (even a single HO cut) and radiation/radiomimetics would be ideal.

    5. OPTIONAL: Given the lack of the G2/M arrest when Cas9 is expressed in asynchronous cultures, the authors may try to synchronize cells in G1 and G2/M before Cas9 is induced. This could help them find out if the G1 and G2/M peaks in Figure 2A are caused by DSBs leading to both a G1 and a G2/M arrest. Alternatively, they could film the cells after Cas9 is added and check microcolony formation.

    6. The timeframes of Rad52 foci in Figure 5 indicate an anomalous pattern, which raises concerns about the validity of the experiment setup. The selected cells did not change their morphology throughout the 140-minute observation period. The unbudded cell remained unbudded, and the bud did not grow in six out of seven cells. For cells with small buds (cells 2 to 5), the expectation was that the bud would grow until the cell either became a dumbbell (indicative of a G2/M arrest) or divided its nucleus. Furthermore, no re-budding events were observed. Is this pattern real? Why is it so? Once this issue is addressed, could the duration of the Rad52 foci be quantified? Was a single z-plane taken? If so, some Rad52 foci that appear and/or disappear could reflect their migration to an on-focus or out-of-focus plane.

    7. OPTIONAL: It would be beneficial to conduct a double labeling with known DSB factors that coexist with Tel1, or are downstream of it, in order to enhance the data shown in Figures 6 and 7. This would also address some of the queries raised about Tel1 clustering and location on the nuclear periphery.

    8. In the experiment with the condensin mutant smc2-8, a temperature shift from 24 to 37 ºC is carried out, which represents a significant physiological change. Therefore, it is essential to include a parallel control with a WT strain to rule out the possibility that the unclustering of Tel1 foci is due to the temperature shift.

    9. The sentence in the Discussion about condensin's role in maintaining Tel1 clustering is misleading. It could be interpreted as suggesting that condensin actively gathers Tel1 foci after DSBs (lines 650-651), when in fact condensin's function is simply to maintain Ty element clustering prior to DSBs, as the authors themselves cite in the text.

    10. Taking into account the importance of Cas9/sgRNA plasmid constructions, PFGE and Southern blot in this work, the author should make the effort to describe them all in much more detail in M&M.

    Minor comments:

    1. In the Figure 7 legend, panel A is missing, and the text for the other panels is consequently misplaced.

    2. The functional verification of the yEGFP-Tel1 construction (Fig. 6A & B) would be better presented as a supplementary figure. The same rationale applies to Figure 8.

    3. Please include G2/M in the abbreviations.

    4. Please clarify what is meant by "5h40". Is this 5 hours and 40 minutes? If so, please use alternative nomenclature.

    5. Some sections of the text appear superfluous. For example, the definitions of mean, SEM and SD, and the rationale for choosing SEM over SD (lines 211 to 215), as well as the information about the purpose of PFGE in a figure legend (line 322).

    Referees cross-commenting

    After reading the comments of the other reviewers, I agree with them, and some of them raise the same concerns as I do.

    Significance

    General assessment: The study addresses the generation of multiple DSBs by Cas9 when an increasing number of targets are incorporated for cutting. The strategy to create an increasing dose of DSBs based on the different Ty elements is an innovative approach, although it is constrained by the nature of the target. The assessment of DSBs by PFGE plus Southern blot is well planned, although there is potential to exploit the obtained results further to assess the actual vs theoretical DSBs, saturation effect, etc. They then sought to use their dose-dependent system to examine the role of Tel1 in the DSB response, comparing Cas9 with zeocin. However, a comparison between the two strategies is challenging without prior knowledge of the number of DSBs present in each treatment. Overall, the study represents a promising effort to use Cas9 for the generation of multiple DSBs in yeast. Nevertheless, the system is constrained for further mechanistic studies on the DSB response due to its slow kinetics, low yields, and lack of expected DNA damage responses, particularly the G2/M checkpoint.

    Advance: Despite such a dose-response assessment for the Cas9-based DSBs have not been performed in yeast, similar studies with sequence-specific DSBs have been done before in Lorraine Symington's lab (and others). Perhaps the Cas9-based system is simpler than the one that relies on multiple insertions of the HO cutting sequence, but it appears neither simpler than the one based on the expression of restriction enzymes nor more efficient. The Cas9 system has several limitations that make it less suitable for studying how cells react against multiple DSBs. It is slow, probably saturates after a few DSBs, and does not render a full DNA damage response. However, there is still value in understanding and making predictions about this important gene-editing method.

    Audience: This paper is intended for a specialized audience, including those involved in basic research on DSB sensing and repair, particularly in yeast.

    Field of expertise of the reviewer: Cell and molecular biology of S. cerevisiae, with a particular interest in the DNA damage response.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary.

    The authors have developed a system to generate a variable number of double-strand breaks (DSBs) - specifically 1, 15, or 59 - in the genome of Saccharomyces cerevisiae in a galactose-inducible manner. This system relies on the galactose-inducible expression of Cas9 nuclease and the use of guide RNAs (gRNAs) targeting different classes of Ty elements. The efficiency of DSB induction was assessed through several methods, including monitoring cell viability in media containing galactose versus glucose, pulsed-field gel electrophoresis (PFGE), PFGE combined with Southern blotting using probes specific to different chromosomes, and the formation of foci by repair and checkpoint proteins. Specifically, the system was utilized to examine foci of the apical checkpoint kinase Tel1 and the repair factors RPA and Rad52.

    From their experiments, the authors conclude that the system is capable of inducing DSBs in a controlled and dose-dependent manner, and that the DSBs are indeed induced at the chromosomal loci where the Ty elements reside.From microscopy experiments, the authors conclude that some DSBs are more persistent, while many are repaired via homologous recombination (HR). New DSBs can form as a result of the continuous expression of Cas9. Finally, Tel1 appears to form multiple foci after DSB induction and these foci localize to the nuclear periphery.

    Major comments.

    In my opinion, some experiments lack appropriate quantification, particularly those evaluating the dose-response effect and the efficiency of DSB induction. Additionally, some conclusions appear to be overestimated in relation to the experimental data. Further experiments and more detailed analyses would be beneficial to fully support the claims and conclusions presented. The data and methods are presented in a manner that should allow for reproducibility. The experiments are adequately replicated, but the statistical analyses are lacking in most of the figures. For example, in Figures 4B, 4C, and 6D, it would be helpful to indicate when the increase in cells containing repair foci is significant.

    Additional experiments and modifications suggested:

    • Figure 1B: Quantify the effect of DSB induction on cell viability with a dose-response curve. No differences are observed between the 0.5% gal and 1% gal conditions. The greatest effect on viability occurs in 2% gal and in the absence of sucrose, which might be due to the absence of sucrose. This should be addressed or discussed.
    • Figure 1B and Figure 2: A quantification of galactose-induced Cas9 expression in the presence of different doses of galactose or over time, respectively, is a necessary control (mRNA or protein expression).
    • Figure 2A: From the cell cycle analysis using FACS, the authors suggest that Cas9 induction causes checkpoint activation. This can be easily confirmed by using more direct methods for checkpoint activation, such as Rad53 phosphorylation. In addition, checkpoint activation should be monitored in synchronized cells released in galactose in order to evaluate whether Cas9-induced DSBs in Ty elements can trigger checkpoint in the first cell cycle or require more time. This reflects the timing of DSB induction.
    • Figure 3: In addition to the restriction analysis, kinetics of DSB formation at specific Ty loci by classical Southern blot or qPCR is, in my opinion, necessary to demonstrate the effectiveness and efficiency of DSB induction over time, especially in relation to Cas9 expression.
    • Figure 5: In this case as well, I believe that a quantitative analysis of the number of cells in the different conditions illustrated in the figure is necessary to understand the dynamics of repair and the formation of new DSBs. Some conclusions are somewhat strong, particularly the correlation between cell cycle phase and repair kinetics (long-lasting Rad52 foci versus short-lived Rad52 foci) and would require quantitative and statistical analysis. In addition, it is not clear to me why cells in which the Rad52 focus disappears do not proceed through the cell cycle (e.g., cells represented in rows 8 and 9 of the figure).
    • Figure 7E and F: It would be interesting to see if the number of Rad52 foci per cell changes in the smc2-8 mutant to understand if RAD52 is required to form the repair center or if this depends on the clustering of Ty elements at the nuclear periphery.

    Minor comments.

    The authors have cited relevant literature to support their methodology, findings, and conclusions. The text and figures are clear. The descriptions in the text are precise and well-articulated, making the data easy to understand. The figures are well-designed and clearly labeled.

    Specific suggestions:

    • Figure 2C: Please change the order of the panel: place chromosome III at the top and chromosome IV at the bottom, as described in the text.
    • Figure 4: The dose-response effect is not evident when monitoring repair foci. It appears that the proportion of cells showing RPA and Rad52 foci is generally low, especially after the formation of 59 DSBs. This is particularly concerning given that the strain in which 59 DSBs are induced already has 20% of cells with RPA foci at time = 0. The authors attribute the lower presence of foci to improved repair caused by the clustering of multiple lesions, but could it simply be due to lower Cas9 efficiency when there are many target sites?
    • Related to Figure 4:The statement on line 400: "the proportion of nuclei displaying Rfa1 foci was consistently double than that of nuclei bearing Rad52 foci, probably reflecting the increased residence time of resected filaments in comparison with the process of homology search" is somewhat strong, considering that the proteins are labeled with different fluorophores, which might experience different rates of photobleaching.

    I believe the proposed experiments can be completed in about 6 months. The request to monitor the kinetics of DSB formation at specific Ty elements in more detail might take more time if the PCR or Southern blot techniques need to be optimized. However, the authors may have other methods in mind that are more familiar to them for evaluating these kinetics, which could be equally valid.

    Referees cross-commenting

    I have carefully read the comments on the manuscript from the other reviewers. I noticed that many of our opinions coincide, and I am convinced that all the requests are appropriate.

    Significance

    My field of expertise centers on checkpoint regulation and homologous recombination in Saccharomyces cerevisiae. I have extensively utilized the galactose-inducible HO endonuclease system for inducing DSBs. I believe that developing additional systems to induce one or more localized DSBs in specific genomic regions is crucial for addressing unresolved questions regarding DSB response. An ideal system would also operate independently of galactose. Based on my experience, an effective system for DSB induction should induce breaks rapidly and simultaneously to produce innovative and reproducible results. From the data presented, I am uncertain whether the system developed in this work meets these criteria.

    The development of a Cas9-based system capable of forming multiple DSBs could be an important tool for studying the DSB response, although I have doubts about how much it will truly enhance our understanding of damage repair. Other systems, cited by the authors, have been previously developed to produce multiple DSBs with different nucleases and using TY elements. Although the Ty-HO system to induce 1, 7, or 10 DSBs was developed in L. Symington's laboratory in 2004, the use of this system has been very limited, likely because it is not easy to monitor what happens to individual DSBs and because the efficiency of simultaneously inducing multiple DSBs may decrease with the increase in the number of target sites. I am concerned that the tool developed in this research article may have limited applicability, being relevant primarily to a small niche within the scientific community focused on DSB response, and thus might generate only a narrow interest. However, the latter part of the paper, which addresses the localization of Tel1, seems promising, despite being preliminary. Additionally, the development of a fluorescent variant of Tel1 is intriguing. This new variant appears to retain the protein's functions and forms well-visible foci within the cell, which seem to be brighter and more intense compared to those obtained with the variant previously developed by M. Lisby and R. Rothstein.

    Strengths:

    • The study presents an innovative system to induce a variable number of double-strand breaks (DSBs) in the genome of Saccharomyces cerevisiae using a galactose-inducible Cas9 and specific gRNAs for Ty elements.
    • The authors use a variety of methods to evaluate DSB induction, including cell viability assays, PFGE, Southern blotting, and foci formation of repair and checkpoint proteins. This comprehensive approach provides sufficient evidence that DSBs are induced.
    • The latter part of the article, focusing on the formation and localization of Tel1 foci, is particularly important. It sheds new light on the functions of Tel1 and its role in the DSB response. This finding is a significant preliminary indication that warrants further development, as the authors suggest in the discussion. I find the development of a tool to effectively visualize Tel1, which is a low-abundance protein in the cell, to be innovative and important for the community working in DSB repair and checkpoint.

    Limitations:

    • Some experiments lack appropriate quantification. For instance, a dose-response curve quantifying the effect of DSB induction on cell viability is missing. Additionally, quantification of galactose-induced Cas9 expression over time (mRNA or protein) is necessary.
    • Statistical analyses are lacking in most figures. It is important to indicate when increases in cells containing repair foci are significant, particularly in Figures 4B, 4C, and 6D.
    • The suggestion of checkpoint activation from cell cycle analysis using FACS should be validated with more direct methods, such as Rad53 phosphorylation, and monitored in synchronized cells to evaluate the timing of checkpoint activation.
    • Further analysis is needed to demonstrate the effectiveness and efficiency of DSB induction over time, especially in relation to Cas9 expression. Monitoring the kinetics of DSB formation at specific Ty loci by classical Southern blot or qPCR would be beneficial.
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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript entitled "A CRISPR-Cas9-based system for the dose-dependent study of DNA double-strand breaks sensing and repair" by Coiffard and colleagues reports on a novel system to monitor the response to multiple DSBs in yeast by targetting Cas9 nuclease to Ty elements. The data is clearly presented, but there are several technical concerns and questions the conclusions of the manuscript. First of all, it is not clear how many DSB cells contain at any given time. The kinetics of DSB formation appears to be slow (hours) relative to the formation and progression of DNA repair foci (minutes). Tel1 foci are found primarily at the nuclear pheriphery, but given the slow kinetics and low number of foci, it is unclear whether the peripheral localization of Tel1 represents physiologically relevant repair and aberrant events where repair failed. Unfortunately, there is not assessment of the outcome of repair. Is the repair error-free or do the cells accummulate indels and/or structural changes at the Cas9 cleavage sites? Without this information, the assay will be of limited use for the scientific community.

    Other major issues:

    1. The introduction should reflect on the chemical structure of DSB ends and the fact that Cas9 remains bound to DNA after cleavage, which may delay repair.
    2. Different Ty elements may be cut with different kinetics. The authors should compare their system to (Gnügge and Symington 2020).
    3. The introduction should reflect on the physiological relevance of 15/59 DSBs.
    4. What is the copy number of the Cas9 and gRNA plasmids? Could cell-to-cell variation in copy number explain the variation in the number of Tel1.
    5. Do the authors observe mutations caused by leaky expression of gRNAs in the uninduced state?
    6. Line 413: I don't think the data in figure 4 completely warrants the conclusion the "DSBs induced by Cas9 engage into HR in a dose-dependent manner". More foci with 15 DSBs than with 59 DSBs? Interference or other explanations? I think this discrepancy warrants additional discussion.
    7. What is observed with a 1h pulse of Cas9 induction followed by glucose? Can the assay monitor the kinetics of repair?
    8. The interpretation of foci in figure 5 is difficult to follow given that only 1 focus is observed while many DSBs are induced. Without further experimentation, these speculations should be moved to the Discussion.
    9. Tel1 foci were always observed at the nuclear periphery (figure 6C). This information should be quantified and compared to Rad52, given that Rad52 foci have previously also been observed at the nuclear periphery for persistent DNA lesions (Whalen and Freudenreich, 2020; Nagai et al., 2008; Lisby et al., 2010). Do these foci perhaps reflect a small subset of Cas9-induced DSBs that are not repair?
    10. Line 699: the notion that Tel1 senses DSBs is novel and does not fit well with the literature given that Tel1 is recruited to foci downstream of Mre11 (Lisby et al., 2004). Unless the authors can provide additional evidence, I suggest to rather write that Tel1 is an early transducer of the DNA damage response.

    Minor suggestions:

    1. The manuscript could benefit from correction of English grammar.
    2. Figure 7F: please also include cells with 1 focus in the graph.
    3. Figure 7: the labels A-F do not follow the legend.
    4. Figure 3, legend: it should be stated in the legend, how long Cas9 was induced in this experiment.
    5. Line 673: it could be noted that in mammalian cells, many more foci are observed, which is probably due to the larger size of the nucleus.
    6. The conclusion that Tel1 behaves exceptionally in terms of the number of foci that are formed is perhaps an overstatement, since only three proteins were analyzed and the occurrence of 8 foci was rare (1:1000 cells).
    7. Line 685: the word "form" indicates that the DSB are already at the nuclear periphery, when bound by Tel1. How do the authors exclude that Tel1 foci form all over the nucleus and then subsequently relocalize to the nuclear periphery? Time-lapse microscopy would be able to reveal where the Tel1 foci form.

    Referees cross-commenting

    I have read and agree with the comments of the other reviewers.

    Significance

    The manuscript entitled "A CRISPR-Cas9-based system for the dose-dependent study of DNA double-strand breaks sensing and repair" by Coiffard and colleagues reports on a novel system to monitor the response to multiple DSBs in yeast by targetting Cas9 nuclease to Ty elements. The data is clearly presented, but there are several technical concerns and questions the conclusions of the manuscript. First of all, it is not clear how many DSB cells contain at any given time. The kinetics of DSB formation appears to be slow (hours) relative to the formation and progression of DNA repair foci (minutes). Tel1 foci are found primarily at the nuclear pheriphery, but given the slow kinetics and low number of foci, it is unclear whether the peripheral localization of Tel1 represents physiologically relevant repair and aberrant events where repair failed. Unfortunately, there is not assessment of the outcome of repair. Is the repair error-free or do the cells accummulate indels and/or structural changes at the Cas9 cleavage sites? Without this information, the assay will be of limited use for the scientific community.

  5. Version published to 10.1101/2021.10.21.465387v2 on bioRxiv
  6. Version published to 10.1101/2021.10.21.465387v1 on bioRxiv