Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript contains numerous grammatical errors, awkward phrasing, and typographical issues that distract from the scientific content. For example, the abstract reads “by robust, cheap, and convenient way” (lines 31–32), and Section 5.2 includes the repetition “by by reshaping” (line 434). A thorough language revision by a native English speaker or professional editing service is strongly recommended.

2. The review is rich in compiled studies, but it reads more as a catalog than a critical synthesis. Across Sections 3–5 and Tables 1–3, similar genes are listed under different technologies, yet there is little comparative analysis of why certain strategies succeeded or failed. Adding a dedicated synthesis subsection would greatly increase the manuscript’s scientific value.

3. The “sweet spots” concept introduced in Section 6 (lines 533–540, Figure 5) is intriguing, but the claim that these regions are “amenable to precise editing without compromising growth” is not supported by functional validation or editing‑efficiency data. I suggest tempering the language to present this as a hypothesis‑generating observation rather than a demonstrated phenomenon.

4. While Section 8 (lines 696–738) covers off‑target effects and regulatory aspects, critical practical limitations are largely absent. Issues such as low transformation efficiency in elite germplasm, genotype‑dependency of editing success, and the scarcity of long‑term field trials are not discussed. Addressing these would provide a more realistic view of the pathway to commercial application.

5. Sections 7.2 and 7.3 (lines 618–681) discuss integrating pan‑genomics and genomic selection with CRISPR, yet the authors explicitly note that “no concrete experimental evidence” exists for the latter in canola. While forward‑looking perspectives are valuable, a clearer distinction between established examples (e.g., BnaGTR2) and conceptual frameworks would improve balance and credibility.

6. Heavy metal tolerance receives only superficial attention in Section 5.2 (lines 509–524 and Table 3), with just two examples (BnCUP1, BnaNRAMP1). Given the growing importance of heavy metal stress in agriculture, expanding this section to include other metals (e.g., arsenic, lead) or discussing implications for food safety would make the review more comprehensive.

Author Response

Comment 1: The manuscript contains numerous grammatical errors, awkward phrasing, and typographical issues that distract from the scientific content. For example, the abstract reads “by robust, cheap, and convenient way” (lines 31–32), and Section 5.2 includes the repetition “by by reshaping” (line 434). A thorough language revision by a native English speaker or professional editing service is strongly recommended.

Response 1: Grammatical errors and typographical issues have been carefully checked and corrected in the whole text.

Comment 2. The review is rich in compiled studies, but it reads more as a catalog than a critical synthesis. Across Sections 3–5 and Tables 1–3, similar genes are listed under different technologies, yet there is little comparative analysis of why certain strategies succeeded or failed. Adding a dedicated synthesis subsection would greatly increase the manuscript’s scientific value.

Response 2: 

We understood the concerns regarding Sections 3–5 and Tables 1–3. We revised to keep these sections as concise as possible while still providing sufficient context for the studies discussed. In addition, after Sections 3–5, we added a NEW synthesis section titled “6.0 Insights from Cross-Technology Gene Manipulation”.

This section summarizes and integrates the key findings across the different approaches discussed (genetic modification, RNAi, and genome editing), highlighting the common biological mechanisms underlying stress adaptation. The detailed examples presented in Sections 3–5 are also complemented by Tables 1–3, which provide a concise overview of the key genes, strategies, and outcomes reported in the literature.

We believe that this structure allows readers to first examine the specific studies within each technology and then refer to the synthesis section for a broader comparative perspective. This approach helps reduce redundancy while preserving the important details needed to understand the biological context and implications of the reported findings.

Comment 3. The “sweet spots” concept introduced in Section 6 (lines 533–540, Figure 5) is intriguing, but the claim that these regions are “amenable to precise editing without compromising growth” is not supported by functional validation or editing‑efficiency data. I suggest tempering the language to present this as a hypothesis‑generating observation rather than a demonstrated phenomenon.

Response 3: Yes, the term “sweet spots” could overstate the current evidence. So, the text to describe these regions were changed into “prospective sites”, highlighting them as promising candidates for genome editing while maintaining a hypothesis-generating framing that remains to be validated. We added these sentences, shown in lines 729 to 732, “These physically clustered genes may therefore represent prospective sites for targeted modification, highlighting promising candidates for future genome editing, although this hypothesis-generating observation remains to be validated.”

Comment 4. While Section 8 (lines 696–738) covers off‑target effects and regulatory aspects, critical practical limitations are largely absent. Issues such as low transformation efficiency in elite germplasm, genotype‑dependency of editing success, and the scarcity of long‑term field trials are not discussed. Addressing these would provide a more realistic view of the pathway to commercial application.

Response 4: In the revised version we added a new paragraph, shown in lines 871 to 880, to address these and to provide a more realistic view of the pathway to commercial application: “Beyond genomic precision, significant practical limitations also hinder the commercial pipeline of gene edited B. napus. The most important constraint is the low efficiency of transformation, and high recalcitrance of elite germplasm [233]. While lab scale experiments in model cultivars such as 'Westar' may achieve laboratory success, there remains a strong genotype dependence for regeneration and editing that limits direct improvement of high-yielding commercially important cultivars that have been adapted to local environments over many years [234,235]. There is also a lack of longer-term field trials to assess the stability of edited traits, particularly complex multigenic traits such as heat and drought tolerance that are to be tested under variable and often unpredictable agro-climatic conditions for realistic commercial application [236].”

Comment 5. Sections 7.2 and 7.3 (lines 618–681) discuss integrating pan‑genomics and genomic selection with CRISPR, yet the authors explicitly note that “no concrete experimental evidence” exists for the latter in canola. While forward‑looking perspectives are valuable, a clearer distinction between established examples (e.g., BnaGTR2) and conceptual frameworks would improve balance and credibility.

Response 5: We agreed that Sections 7.2 and 7.3 (Sections 8.2 and 8.3 in the revised version) could benefit from a clearer distinction between established examples and conceptual frameworks, and we revised the text as below:

(1) Highlight BnaGTR2 as an established example of pangenome-guided CRISPR validation in canola.

We’ve added a sentence like this to highlight the BnaGTR2, see in lines 793-794 “A well-characterized example in canola involves the glucosinolate transporter BnaGTR2. “

(2) Clearly mark genomic selection (GS)-guided CRISPR in canola as hypothetical/ conceptual, ensuring forward-looking perspectives are distinguished from experimental evidence.

In Section 8.3, we explicitly note that GS-guided CRISPR integration is currently hypothetical in canola, describing it as a conceptual, forward-looking approach. We have emphasized terms such as “no concrete experimental evidence,” “potential,” “currently hypothetical,” and “in principle” to ensure the distinction is clear.

The version for now is shown in lines to 820 -833:

“8.3. Intersection of CRISPR and genomic selection

Currently, there is no concrete experimental evidence of genomic selection (GS) predictions being directly validated by CRISPR within the same breeding program. However, several reviews have discussed the potential of integrating these approaches [212-214]. In these frameworks, GS provides a genome-wide predictive model to identify promising alleles and elite genetic backgrounds, which CRISPR could then edit to functionally validate predictions and utilize available genetic resources for crop improvement [215].

While this approach is currently hypothetical in canola, GS-guided CRISPR could, in principle, be applied to precisely edit key genes associated with yield, stress tolerance, and quality traits, accelerating the development of superior varieties while maintaining favorable genetic backgrounds and maximizing the use of existing genetic diversity.”

We’ve also reflected this to the abstract.

Comment 6. Heavy metal tolerance receives only superficial attention in Section 5.2 (lines 509–524 and Table 3), with just two examples (BnCUP1, BnaNRAMP1). Given the growing importance of heavy metal stress in agriculture, expanding this section to include other metals (e.g., arsenic, lead) or discussing implications for food safety would make the review more comprehensive.

Response 6: We agreed and added a new paragraph as shown in Lines 629-637 in the revised version:

“CRISPR/Cas-mediated improvement of heavy metal tolerance in B. napus remains largely limited to cadmium-focused studies. Other heavy metals, such as arsenic and lead are also a serious threat to canola yield and quality. But gene editing approaches are lacking to target these genes so far. Several studies have been conducted in other plant species that target genes involved in metal transport and detoxification, including heavy metal ATPases (HMAs) [171] [253], OsPMEI [172] [254], OsLsi1, OsLsi2 and OsNIP3;1 [173] [174], which play critical roles in regulating the uptake, sequestration, and detoxification of multiple heavy metals. Therefore, these genes could be promising targets for future CRISPR/Cas-based engineering in B. napus.”

Reviewer 2 Report

Comments and Suggestions for Authors

A well structured and rigorous review effectively integrates the latest advances in gene editing (GM), RNA interference (RNAi), and CRISPR to enhance the resistance of Canola to biotic and abiotic stresses. It provides comprehensive and up-to-date insights into the field, making valuable and timely contributions to current research

1. Lines 152–156: The long sentenceis acceptable but could be split after “every crop.” for better readability.
2. Tables 1–3 Formatting consistency throughout Tables 1 (lines 215–216), 2 (lines 326–327)and 3 (lines 523–524), Gene symbols are not uniformly italicized Pls fix this issue (e.g., BnaWRKY75, BnF5H), and also minor typos in donor names (e.g., “T. atroviride” should be consistently formatted). Apply uniform italicization and check alignment in final version.
3. Figure 5 (line 543) italicize Brassica napus and ensure the proper formatting for species names in whole MS.
4. Line 434, I suggest author should also insert this citation “Wang, M.; Du, P.; Xi, L.; Lin, H.; Zhang, S. Dynamic Coordination: How ERF Transcription Factors Coordinate Plant Development and Adaptive Stress Responses. Biomolecules 2026, 16, 466. https://doi.org/10.3390/biom16030466”
5. Ensure all gene names (BnaABI5, BnVTC, BnaC07.GLIP1, etc.) are consistently italicized, e.g. 104–105, 178–179, 267–270, 552–553
6. Lines 139–141, I suggest author should also insert this citation, Xie, X.; Hu, Y.; Li, X.; Li, S.; Li, X.; Li, Y. Measuring and Enhancing Food Security Resilience in China Under Climate Change. Systems 2025, 13, 1054. https://doi.org/10.3390/systems13121054
7. The author thoroughly review the manuscript for grammatical errors and make necessary corrections throughout the entire MS.

Author Response

Comment 1: Lines 152–156: The long sentenceis acceptable but could be split after “every crop.” for better readability.

Response 1: Agreed and revised accordingly.

Comment 2: Tables 1–3 Formatting consistency throughout Tables 1 (lines 215–216), 2 (lines 326–327)and 3 (lines 523–524), Gene symbols are not uniformly italicized Pls fix this issue (e.g., BnaWRKY75, BnF5H), and also minor typos in donor names (e.g., “T. atroviride” should be consistently formatted). Apply uniform italicization and check alignment in final version.

Response 2: Agreed and corrected accordingly.

Comment 3: Figure 5 (line 543) italicize Brassica napus and ensure the proper formatting for species names in whole MS.

Response 3: Agreed and revised accordingly.

Comment 4: Line 434, I suggest author should also insert this citation “Wang, M.; Du, P.; Xi, L.; Lin, H.; Zhang, S. Dynamic Coordination: How ERF Transcription Factors Coordinate Plant Development and Adaptive Stress Responses. Biomolecules 2026, 16, 466. https://doi.org/10.3390/biom16030466”

Response 4: Added in the revised version

Comment 5: Ensure all gene names (BnaABI5, BnVTC, BnaC07.GLIP1, etc.) are consistently italicized, e.g. 104–105, 178–179, 267–270, 552–553

Response 5: Agreed and corrected accordingly.

Comment 6: Lines 139–141, I suggest author should also insert this citation, Xie, X.; Hu, Y.; Li, X.; Li, S.; Li, X.; Li, Y. Measuring and Enhancing Food Security Resilience in China Under Climate Change. Systems 2025, 13, 1054. https://doi.org/10.3390/systems13121054

Response 6: Added in the revised version.

Comment 7: The author thoroughly review the manuscript for grammatical errors and make necessary corrections throughout the entire MS.

Response 7: Grammatical errors and typographical issues have been carefully checked and corrected in the whole text.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

It is worth noting that you have included the latest genetic engineering approaches in their review, highlighting the fields of pangenomics, AI integration and in silico off-target detection tools.

Overall, the review is well written and could be used as high-quality educational material for university students.

However, I feel that some sections are overly lengthy, particularly Chapters 3, 4 and 5. While I understand the authors' intent, I believe that a brief introduction to each chapter followed by a tabular summary of the key information with references would sufficent. Furthermore, the additional information is an unnecessary duplication of subsections 3.1–3.2, 4.1–4.2 and 5.1–5.2.

Tables 1–3 require formal revision: merge cells representing a single observed factor and ensure the table header is included on subsequent pages. Full Latin species names must be provided in the author citations. Consider the label for the 'stress name' column. It would be better to use a more scientific-sounding label, such as 'abiotic/biotic stress factor'.

In accordance with official botanical nomenclature, all Latin species names and author abbreviations must be written in italics throughout the manuscript. Similarly, Latin family names should be written in italics.

In the introductory sections of the manuscript, please clearly specify the difference between rapeseed and canola. Alternatively, move paragraph 57–60 to an earlier part of the manuscript. Figure 1 is missing the name of the crop whose growth it depicts. References must be included in Figure 2, as it presents specific percentage data. You must also cite the sources of the images used to create Fig. 2.

Please also provide the Latin names of the diseases and pests (lines 115–121). The description of Figure 4 must also include the symbols “a–j” as marked in the figure.

Specify the type of tool used to generate Figure 5. The title of Figure 6 appears three times. The caption for this figure must include explanations of any abbreviations used.

It is necessary to disclose the use of AI or any other tool used in the creation of images and individual pictograms.

Formatting errors: row 38 - references

 

Author Response

Comment 1: It is worth noting that you have included the latest genetic engineering approaches in their review, highlighting the fields of pangenomics, AI integration and in silico off-target detection tools. Overall, the review is well written and could be used as high-quality educational material for university students.

Response 1: Thanks so much for these encouraging comments.

Comment 2: However, I feel that some sections are overly lengthy, particularly Chapters 3, 4 and 5. While I understand the authors' intent, I believe that a brief introduction to each chapter followed by a tabular summary of the key information with references would sufficent. Furthermore, the additional information is an unnecessary duplication of subsections 3.1–3.2, 4.1–4.2 and 5.1–5.2.

Response 2: We understood the concerns regarding Sections 3–5 and Tables 1–3. We revised to keep these sections as concise as possible while still providing sufficient context for the studies discussed. In addition, after Sections 3–5, we added a NEW synthesis section titled “6.0 Insights from Cross-Technology Gene Manipulation”.

This section summarizes and integrates the key findings across the different approaches discussed (genetic modification, RNAi, and genome editing), highlighting the common biological mechanisms underlying stress adaptation. The detailed examples presented in Sections 3–5 are also complemented by Tables 1–3, which provide a concise overview of the key genes, strategies, and outcomes reported in the literature.

We believe that this structure allows readers to first examine the specific studies within each technology and then refer to the synthesis section for a broader comparative perspective. This approach helps reduce redundancy while preserving the important details needed to understand the biological context and implications of the reported findings.

Comment 3: Tables 1–3 require formal revision: merge cells representing a single observed factor and ensure the table header is included on subsequent pages. Full Latin species names must be provided in the author citations. Consider the label for the 'stress name' column. It would be better to use a more scientific-sounding label, such as 'abiotic/biotic stress factor'.

Response 3: Agreed and revised accordingly.

Comment 4: In accordance with official botanical nomenclature, all Latin species names and author abbreviations must be written in italics throughout the manuscript. Similarly, Latin family names should be written in italics.

Response 4: Agreed and revised accordingly.

Comment 5: In the introductory sections of the manuscript, please clearly specify the difference between rapeseed and canola. Alternatively, move paragraph 57–60 to an earlier part of the manuscript. Figure 1 is missing the name of the crop whose growth it depicts. References must be included in Figure 2, as it presents specific percentage data. You must also cite the sources of the images used to create Fig. 2.

Response 5: Agreed and revised accordingly.

Comment 6: Please also provide the Latin names of the diseases and pests (lines 115–121). The description of Figure 4 must also include the symbols “a–j” as marked in the figure.

Response 6: Agreed and revised accordingly.

Comment 7: Specify the type of tool used to generate Figure 5. The title of Figure 6 appears three times. The caption for this figure must include explanations of any abbreviations used.

Response 7: Agreed and revised accordingly.

Comment 8: It is necessary to disclose the use of AI or any other tool used in the creation of images and individual pictograms.

Response 8: draw.io (https://app.diagrams.net) online tool used to make figures

Comment 9: Formatting errors: row 38 - references

Response 9: Agreed and revised accordingly.