Genome-Wide CRISPR Screens Reveal ZATT as a Synthetic Lethal Target of TOP2-Poison Etoposide That Can Act in a TDP2-Independent Pathway

Round 1

Reviewer 1 Report

In this manuscript the authors suggested that ZATT is a critical determinant that dictates responses to ETO treatment and targeting ZATT is a promising strategy to increase ETO efficacy for cancer therapy. For example, genome-wide CRISPR screens revealed that ZATT also has a TDP2-independent role in promoting cell survival following ETO treatment. The authors confirmed that ZATT KO cells showed relatively higher ETO sensitivity than TDP2-KO cells, and ZATT/TDP2 DKO cells displayed additive hypersensitivity to ETO treatment. Depletion of ZATT resulted in accelerated TOP2 degradation after ETO or cycloheximide treatment, suggesting that ZATT may increase TOP2 stability and likely participate in TOP2 turnover. The manuscript is well and carefully written, data are solid and conclusions are justified by the results. The discussions on the primary results and their significance are scientifically sound.

I recommend to accept Ms in the current form.

Author Response

Thanks for your support of our manuscript! 

Best,

Junjie

Reviewer 2 Report

• Overall this article is good to publish with minor revisions.
• Review was nicely designed and presented well. Methods are clearly explained, detail description provided in general and gradually going into specific details.
• Each section is demonstrated with adequate details.
• Appropriate references were cited.
• Abbreviations elaboration in beginning is recommended to remind the reader in the beginning of the article. 
• Conclusion part can be more specific with additional detailing with respect to the future directives. 
• It is recommended to use 'this review or in this work' instead of using 'we' throughout the review.
• doi is missing for the reference 4.
• In line 107 reference should be corrected as 12-15 instead of 12,15.
• It is recommended to have discussion part at the end of the article after materials and methods for a better understanding of the readers.
• Combining both the supplementary data and providing description of the graphs and data will help the readers to follow easily (Although presented in the actual article).

Author Response

Thanks for the nice comments. Please see below for the point-by-point response to the reviewer's comments.

Best,

Junjie

• Abbreviations elaboration in beginning is recommended to remind the reader in the beginning of the article. 

We have added explanations for the abbreviations at the beginning of the article

• Conclusion part can be more specific with additional detailing with respect to the future directives. 

We agree that our conclusion can be more specific with additional details regarding future directives. In light of this, we have revised the discussion section to provide future directions. Specifically, we have suggested to introduce the degradation tag (dTAG) system to investigate ZATT-mediated TOP2 regulation. Moreover, we have highlighted the need for additional CRISPR screens and FACS-based screens in ZATT/TDP2 DKO cells to search genes and proteins that are important for TOP2-induced DNA damage signaling beyond ZATT and TDP2.

• It is recommended to use 'this review or in this work' instead of using 'we' throughout the review.

We have replaced instances of "we" with “in this study" or "in this work".

• doi is missing for the reference 4.

We have now included the DOI for reference 4 in the article.

• In line 107 reference should be corrected as 12-15 instead of 12,15.

We would like to clarify that references 12 and 15 (12,15) were intentionally cited separately in line 107, rather than as a range from 12 to 15 (12-15). We believe this citation style better highlights the specific sources used to support our arguments.

• It is recommended to have discussion part at the end of the article after materials and methods for a better understanding of the readers.

We appreciate your suggestion to place the discussion section at the end of the article, but we followed the guidelines provided by the journal for structuring our article and listing the table of contents. As per the journal's instructions, the materials and methods section is placed after the discussion section.

• Combining both the supplementary data and providing description of the graphs and data will help the readers to follow easily (Although presented in the actual article).

We have added a figure legend to the supplementary data.