PONE-D-21-10318

A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station

PLOS ONE

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This study was funded by Boeing (http://www.boeing.com/) and miniPCR bio (https://www.minipcr.com/). E.A.S., E.J.G., and S.K. are employed by miniPCR bio. D.S.C. and K.D.F. are employed by Boeing.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Reviewer #1: 

In the article “A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station” the authors report on a study on the DNA double strand break repair pathway selection in yeast on board the international space station using the CRISPR Cas9 system. Homologous Recombination is discriminated from Non-homologous end joining by next generation sequencing of the DSB site following DSB generation by the CRISPR Cas9 nuclease in the presence of an alternative homologous repair template. The experiments are accompanied by similar experiments conducted on earth to access differences in micro-gravity environment.

Overall the study is sound and demonstrates the future possibilities of molecular biology experiments on board the ISS. Due to the limitations in replicates the scientific outcome is limited. Nevertheless, considering the experimental limitations during space flight, I support the publication of the manuscript and only have some minor points.

Minor points:

Supplementary File naming as well as the authors list is not consistent with the main title.

Introduction, line 38 onwards: The authors should also mention the special type / complexity of DSBs generated by cosmic irradiation present in space.

Introduction, line 55 onwards: The authors should also include cell cycle specific aspects, when they discuss pathway selection DSB repair, especially in humans.

Results, line 103: I would replace he term “ADE2 mutant lines” with “ADE2 mutant colonies”

Results Figure 2: Since the colonies are not clearly visible (at least in the images provided to me) I would suggest to included a cropped inset of one colony each.

Methods, line 297: The authors should add the wavelength at with the O.D. was measured.

Methods, line 301-302: “Final cell pellets..” In my understanding the number of 1x10^8 cells is per pellet or total number, but not cells/ml. Or is this number representing the cell concentration after resuspension/reconstitution? The authors should clarify this point.

Methods, line 329 onwards: The authors describe that both the ground control as the in flight petri dishes were kept at room temperature. RT is not defined and should be given. Was it identical on the ISS and on the ground? If not this could explain the differences in colony size.

Methods, line 341: The bar-coding process should be explained in more detail, may be also with a small sketch, to help the less experienced reader to understand it.

Figure 1 has some elements at the end of the page that are probably misplaced.

Supplementary information: it reads “…Archive (ENA) under project PRJEB39039 (add link)”. No link is available and I could not find the mentioned project in ENA.

Reviewer #2: 

The manuscript by Stahl-Rommel and colleagues report the first attempt to undertake a simple DNA repair assay in space. This is important since an understanding of differences in maintaining genome stability on space have far-reaching implications for technologies that can employed in space and human health in space.

Sensibly, the team opted to use budding yeast as this is the most robust and best-characterised eukaryotic system available, coupled to an assay which suitable for studying the repair of targeted DNA double-strand breaks. The work is clearly presented and the caveats in interpreting the limited data obtained openly discussed.

I have only very minor comments:

1. I would suggest growing the yeast cells to a density of less than 1 x 108 CFU/mL (perhaps 2 x 107) might give a better transformation efficiency in future studies.

2. In the Introduction (p2, line 51) the possibility that microgravity conditions might influence DNA repair pathway usage or efficiency, which is an interesting possibility. It is also worth pointing out to readers that the ionising radiation background in space might damage DNA breaks close to their termini and this could influence repair efficiency. This is especially relevant during repair by HR, where tracts of ssDNA are generated as intermediates that might be more susceptible to such collateral damage. Such 'collateral' damage could plausibly influence the efficiency and outcome of repair.

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Reviewer #1: No

Reviewer #2: Yes: Peter J.McHugh

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A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station

PONE-D-21-10318R1

Dear Dr. Kraves,

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Kind regards,

Ruslan Kalendar, PhD

Academic Editor

PLOS ONE