-->PONE-D-25-53201-->-->CRISPR/Cas9-Mediated Editing in FAD2 Gene to Enhance Oil Quality in Soybean [Glycine max (L.) Merrill]-->-->PLOS One

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Reviewer #1: Partly

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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Reviewer #1: This study addresses a critical agricultural need for improving oil quality in Indian soybean cultivars by applying CRISPR/Cas9-mediated editing of the FAD2 gene. It represents the first report of targeted FAD2 modification in an Indian soybean genotype (GJS-3), successfully enhancing oleic acid content, reducing linoleic acid levels, and improving oil oxidative stability. The research design is comprehensive, encompassing genotype screening, in vitro regeneration system establishment, CRISPR vector construction, Agrobacterium-mediated transformation, and multi-level validation (molecular and phenotypic). This work offers a valuable technical framework and germplasm resource for soybean quality breeding in India, with both theoretical innovation and practical application potential. However, I some concerns listed below before accpeting this manuscript.

In Introduction section

1. Citation Accuracy: Multiple citation errors occur (e.g., "Du et al.11", "Michno et al.12") with no corresponding entries in the reference list (only 10 references are implied, missing entries for 11–13). Complete reference details must be supplemented, and citation formatting should be standardized (e.g., APA or journal-specific style).

2. Strengthen Research Gap Elaboration: While the study mentions "untested Indian germplasm", it lacks specific data on the unique characteristics of FAD2 gene sequences and fatty acid profiles in Indian soybean cultivars (compared to North American/East Asian varieties). This weakens the rationale for selecting GJS-3 as the editing target. Relevant data or literature should be added to justify the specificity of GJS-3 as an ideal candidate.

3. Rigorous Technical Comparison: The claim that "CRISPR/Cas9 is simpler than ZFNs and TALENs" is overly generalized. The comparison should be contextualized to soybean transformation (e.g., citing soybean-specific applications, efficiency differences of ZFNs/TALENs) or simplified to highlight optimizations in the current study (e.g., gRNA specificity, vector selection advantages).

In Materials and Methods section

1. Missing Key Experimental Parameters:

1.1 Fatty acid analysis: The use of "15 g freeze-dried samples" is unusually large (conventional soybean fatty acid analysis requires only tens of milligrams). Details such as dehulling status and grinding particle size (e.g., sieve mesh) must be supplemented to ensure reproducibility of extraction efficiency.

1.2 CRISPR vector construction: No method for gRNA specificity validation is described (e.g., BLAST against the soybean genome to exclude off-target sites). While "specificity score" is mentioned (Supplementary Table S3), the evaluation criteria (e.g., number of off-target sites, homology threshold) should be briefly explained in the main text.

1.3 Agrobacterium transformation: An OD₆₀₀ of 1.0 is excessively high (soybean transformation typically uses OD₆₀₀ = 0.4–0.6). The rationale for this concentration (e.g., pre-experiment optimization results) must be justified to address concerns about contamination risk.

2. Supplementary Experimental Design Details:

2.1 In vitro regeneration: "3 biological replicates per treatment, 10 explants per replicate" requires clarification on whether replicates represent independent culture batches (e.g., seeds from different sowing batches) to avoid statistical bias.

2.2 Molecular detection: The design basis for Cas9 and U3 primers (e.g., targeting conserved vector regions) is not specified. Specificity validation results (e.g., absence of non-specific bands) should be supplemented.

3. Refined Economic Analysis Methods:

3.1 The claim of "processing cost savings of US$10–80 per tonne" requires specific data sources (e.g., UNIDO/AOCS report year, chapter) and should incorporate regional parameters (e.g., local energy prices, labor costs in India) to enhance relevance.

3.2 The market premium of "US$0.05–0.25 per lb" needs temporal and geographical context (e.g., Indian domestic vs. international markets) to avoid ambiguity.

In Result section

1. Data Presentation and Statistical Optimization:

1.1 Tables 1–3 mention "significant differences" but lack specific statistics (e.g., F-values, p-values). Detailed ANOVA results should be supplemented (e.g., in table footnotes). Figures 4–6 should include error bars (standard deviation/standard error) to visualize data dispersion.

1.2 Fatty acid composition: Elaidic acid (C18:1 n-9 trans) is typically <1% in wild-type soybean, but the reported range (191.6–318.6 g kg⁻¹, ~19–32%) is anomalous. This may reflect detection errors (e.g., incomplete separation of cis/trans isomers) or misnaming (e.g., confusing cis-oleic acid with trans-elaidic acid). Results must be revalidated, and fatty acid nomenclature or data corrected.

2. Deepened Mutation-Phenotype Correlation:

2.1 The impact of mutations (T→C, A→C) on FAD2 protein structure/function (e.g., amino acid substitutions, active site disruption) is not analyzed. Bioinformatics analyses (e.g., ExPASy for amino acid changes, SWISS-MODEL for protein structure prediction) should be added to clarify the molecular mechanism.

2.2 Generational stability data (e.g., fatty acid profiles, mutation segregation in T1/T2 generations) are missing. Phenotypic tracking across at least two generations is required to verify heritability of edited traits.

3. Off-Target Effect Detection:

3.1 As a precision editing study, off-target validation is absent. Potential off-target sites should be predicted (e.g., using CRISPOR) and verified via PCR sequencing to demonstrate editing specificity.

In Discussion section

1. Precise Result Interpretation:

1.1 The claim of "transgene-free edited lines" is supported only by PCR, which cannot rule out fragmented integration or copy number variation. Southern blot analysis should be supplemented to confirm the absence of foreign DNA.

When discussing "pleiotropic effects on agronomic traits", the study merely states "future evaluation is needed". Relevant literature (e.g., FAD2 mutations in other crops affecting yield/stress resistance) should be cited to propose specific follow-up research directions.

2. Strengthened Comparison with Similar Studies:

2.1 Key metrics (editing efficiency, oleic acid enhancement, transformation rate) should be compared with other soybean FAD2 editing studies to highlight innovations (e.g., Indian cultivar adaptation, transgene-free editing, high-efficiency regeneration).

2.2 Limitations of conventional breeding for high-oleic soybean (e.g., long breeding cycles, linkage drag) should be discussed to emphasize the advantages of CRISPR technology.

Others

1. Citation Consistency: Citation numbering is inconsistent (e.g., missing entries for 11–13), and duplicate citations occur. References should be standardized, and missing entries supplemented.

2. Figure/Table Standardization:

Supplementary figures/tables (S1–S13, S1–S11) are mentioned but not explicitly cited in the main text (e.g., "see Supplementary Figure S1a–d for fatty acid extraction workflow"). Citations should be supplemented.

Legends for Figures 9a–b, 10, 11 are overly brief. Lane annotations (e.g., "M: 1 kb Marker; WT: Wild type; T3: Edited line T3") and mutation site positions (e.g., base position relative to ATG start codon) should be clarified.

3. ppm is not an international standard unit, the authors should be avoided.

Reviewer #2: Comments to author

The manuscript presents a timely study on improving soybean oil quality using CRISPR/Cas9 editing of the FAD2 gene in an Indian cultivar (GJS-3). The work is novel for Indian germplasm and integrates molecular, biochemical, tissue-culture, and analyses which demonstrates the production of transgene-free edited lines with improved fatty acid profiles.

Points need to be addressed:

• Abstract is too long, concise it with important results of the study.

• Concise the introduction part also, focus more on research gap in Indian soybean CRISPR research and objectives of the study.

• Transformation efficiency, editing frequency, and the logic behind these calculations must be explicitly described and not mixed together.

• Include a basic off-target analysis or discuss the lack of such analysis as a limitation.

• Discussion on the lacks about mechanism of FAD2 mutation consequences.

• Focus the discussion part about interpretation of fatty acid results, comparison on previously edited FAD2 lines, implications on Indian soybean breeding, limitations and future directions of this study.

• All the figures need more clarity.

• Ensure all figures, tables, and supplementary datasets are submitted, properly formatted, and clearly labeled in the whole manuscript.

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Reviewer #1: Yes: Shuijin Hua

Reviewer #2: Yes: Muthukrishnan Arun

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CRISPR/Cas9-Mediated  Editing  in  FAD2 Gene to Enhance OilQuality in Soybean  [ Glycine max (L.) Merrill]

PONE-D-25-53201R1

Dear Dr. Hajare,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Mojtaba Kordrostami, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: (No Response)

Reviewer #2: All the comments raised has been attended by the authors. The current manuscript can now be considered for publication in this journal

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Reviewer #1: Yes: Shuijin Hua

Reviewer #2: Yes: Muthukrishnan Arun

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