Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The role of NRF2 in cancer promotion and drug resistance is an interesting topic. NRF2 primarily functions through activation of antioxidant gene expression, including detoxification enzymes like GSTs, and has a well-established role in protecting cells from highly reactive and redox cycling molecules. Gemcitabine works primarily as a chain-terminating nucleotide analog, so it’s not obvious how NRF2 would affect acute sensitivity to gemcitabine. In this regard, the premise of the study is somewhat doubtful. Gemcitabine has been reported to activate ROS production in pancreatic cancer cells, but this production doesn’t appear relevant to its main anticancer function (PMID 23740244). The results in Fig 2C may reflect this: although most of these CRIPSR sgRNAs are anticipated to reduce NRF2 expression, the effect of the EC50 seems minimal. Determining exactly which CRISPR guides are best for knocking out NRF2 is interesting and may ultimately have clinical relevance, but modulating pancreatic cancer cell sensitivity to gemcitabine is probably not the best place to look.

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Firs, I would like to thank you for such interesting work!

In the manuscript you carefully applied a systematic approach for screening of  multiple sgRNAs targeting distinct functional domains of NRF2 across two PDAC cell lines.

I recommend the manuscript for publication after the following revisions are addressed.

 

Major Concerns

1. Figure panel cross-reference errors.
   Throughout the Results section, figure panel citations appear to be systematically mismatched. Specifically, data described in the text as being shown in "Figure 2B" appears to correspond to Figure 2C, and vice versa. For example, the normalized cell viability dose-response comparisons are described with reference to "Figure 2B," while Figure 2B appears to contain the indel distribution data. The authors are asked to carefully verify and correct all in-text figure panel references before resubmission, as this creates significant confusion for the reader.
2. Inconsistent and incomplete statistical reporting.
   Statistical reporting varies considerably across results. Some comparisons include specific p-values (e.g., p<0.0001, p=0.4862), while others imply statistical significance without reporting a value. The authors are asked to: (a) state explicitly which statistical test was used for each comparison; (b) report the number of independent biological replicates (n) for each experiment clearly in the figure legends; and (c) indicate whether error bars represent SEM or SD. This information is currently absent from most figure legends.
3. Sample size inconsistencies in Figure 2A.
   The legend for Figure 2A reports conflicting n values for the same sgRNA in two separate entries (e.g., "sgRNA 3 n=7" in one entry and "sgRNA 3 n=8" in another). The authors are asked to verify and correct all reported sample sizes to ensure consistency between the figure and its legend.
4. Supplementary Materials placeholder text remains.
   The Supplementary Materials section at the end of the manuscript still contains unedited placeholder text: "Figure S1 title, Table S1 title, Video S1 title." The actual supplementary data are provided in Appendices A1–A4. The authors are asked to either update the Supplementary Materials section to accurately describe the content provided, or reorganize the appendix material into properly numbered supplementary figures with appropriate titles and legends.
5. Absence of western blot quantification.
   While raw western blot images are provided in the appendices (commendably, for transparency), no densitometric quantification normalized to the GAPDH loading control is reported. The authors are asked to include quantified band intensities for NRF2 protein levels across all conditions and sgRNAs tested, to allow objective comparison of the degree of protein knockdown achieved.

 

Minor Concerns

6. Typographical errors.
   The following specific errors should be corrected:

• Abstract, line 23: "dug resistance" should read "drug resistance"
• Lines 191, 268, and 270: "Mia-Paca-22" (with a duplicated "2") should read "Mia-Paca-2" consistently throughout the manuscript
• Line 305: The contraction "it's" is inappropriate in scientific writing; please rephrase (e.g., "it is the disruption of protein function...")

7. Inconsistent gene/protein nomenclature.
   The manuscript alternates between "NRF2" and "Nrf2" without a consistent or explicitly stated convention. The standard nomenclature distinguishes the gene (NRF2, italicized) from the protein (Nrf2, roman type). The authors are asked to apply this convention uniformly throughout the manuscript.
8. Figure 3C — protein structure annotation.
   The SWISS-MODEL protein structure visualization in Figure 3C is a useful conceptual addition, but currently lacks residue numbering or a scale reference. The authors are asked to indicate which specific amino acid residues correspond to the CRISPR cut sites for each sgRNA, or to expand the legend to make this connection explicit.
9. Grammatical issues in the Discussion.
   Several sentences in the Discussion are grammatically incomplete or difficult to parse. For example, the sentence beginning "The dynamic nature of the chromosome and the complex interactions with molecular pathways in each cell type can skew results direct analyses..." (approximately lines 332–334) appears to be missing a verb or conjunction and reads as a run-on. The authors are encouraged to carefully revise the Discussion section for grammatical correctness and sentence-level clarity, and to consider professional English language editing.
10. Abbreviation standardization.
    "INDEL/indel" is used interchangeably throughout the manuscript. The authors should select one style and apply it consistently. Additionally, all abbreviations should be defined at first use in the abstract independently of the main text, as these sections are often read separately.
11. Introduction — incomplete sentence and citation check.
    Line 38 ends with "Making it the 3rd leading cause of cancer related death in the United States," which is a sentence fragment. Please integrate this into the preceding sentence. Additionally, the authors should verify that this statistic is consistent with reference 16 (Siegel et al., 2025 CA Cancer J. Clin.), which is the most recent citation available.

 

In summary, this is a sound and well-structured feasibility study that makes a useful contribution to the field of CRISPR-based combinatorial cancer therapy. The identified issues are largely presentational and correctable without new experimental work. I look forward to reviewing the revised manuscript.

With Kind Regards

Comments on the Quality of English Language

The manuscript is understandable overall, but the English requires moderate improvement before publication. Key issues include: grammatically incomplete sentences in the Discussion (e.g., missing verbs and run-on constructions); use of contractions inappropriate for scientific writing (e.g., "it's," line 305); typographical errors ("dug resistance" in the abstract; "Mia-Paca-22" in multiple locations); redundant and repetitive phrasing in the Results and Discussion; inconsistent hyphenation of compound terms; and occasional missing or incorrect use of articles. The authors are encouraged to seek professional English language editing or review by a native English-speaking colleague prior to resubmission.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The title is scientifically relevant, but it is rather long and somewhat heavy in structure. It would be more impactful if shortened and refocused on the main message, namely NRF2 knockout and restoration of gemcitabine sensitivity in PDAC.

The abstract conveys the general interest of the study, but it contains several linguistic inaccuracies and awkward formulations that should be corrected. It would also be helpful to present the main quantitative findings more clearly and to better define the actual scope of the conclusions.

The introduction is generally well referenced; however, some statements appear overly assertive, particularly when comparing the CRISPR approach with standard anticancer therapies. This section would benefit from more cautious wording and a clearer biological justification for targeting NRF2 in PDAC.

A methodological inconsistency should be clarified regarding the number of guides used. The authors state that eleven gRNA sequences were selected, whereas the Methods section and the reported results appear to correspond to only ten guides.

The nucleofection procedure lacks precision in some important details required for reproducibility. In particular, the post-transfection recovery interval reported as 24–72 h is too broad and should be standardized or justified, as it may influence editing efficiency and downstream functional readouts.

The statistical analysis is not described in sufficient detail in the Methods section. Although several p values and sample sizes are reported in the Results, the statistical tests used, the handling of multiple comparisons, and the distinction between biological and technical replicates should be explicitly stated.

The study is mainly based on bulk edited cell populations, which limits the causal interpretation between genotype and phenotype. The authors themselves acknowledge that clonal isolation will be necessary, and this limitation should be emphasized more clearly in the Discussion.

Several issues of consistency in terminology and formatting should be corrected throughout the manuscript and figure legends. For example, the cell line Mia-Paca-2 is sometimes written as Mia-Paca-22, which creates unnecessary confusion and suggests insufficient editorial revision.

The figures are informative, but some legends remain difficult to follow, especially in the presentation of sample sizes per sgRNA and the organization of the panels. Revising the figure legends to make them more concise, more uniform, and easier to interpret would greatly improve the readability of the results.

Protein-level validation remains essentially qualitative. Since a major part of the interpretation relies on the reduction of NRF2, the authors should consider adding densitometric quantification of the western blots normalized to GAPDH.

The mechanistic demonstration remains incomplete. The introduction highlights downstream NRF2 target genes such as NQO1, HMOX1, and GCLC, yet no downstream marker was experimentally assessed to confirm that NRF2 signaling was effectively inhibited beyond the decrease in protein expression alone.

The Discussion, and especially the Conclusion, are at times too assertive considering the preliminary nature of the study. Expressions such as “There is no doubt” or projections toward clinical development should be toned down, particularly since the supplementary materials still contain placeholders and some formatting issues.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed my recommendations. I recommend the publication of this article in its current form.