Application of Gene Editing Technology in Poultry
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1. page 2, line 40: "with traditional ge" is obviously truncated and should be completely replaced by "with traditional gene editing technologies ..."
2. Page 3, line 105: "The crispr sequence includes a leader region ..." The sentence is slightly lengthy, and it is suggested to split it into short sentences.
3. Page 6, line 145: "Nick ase CAS9 (NCAS9) is generated by deactivating one of the nuclear domains ..." It is suggested to change it to "is generated by activating one of the nuclear domains".
4. In this paper, "gene editing" and "genome editing" are mixed, and it is suggested to unify them into "genome editing" or "gene editing".
5. The names of diseases such as "Avian Leukosis" and "avian influenza" should be capitalized consistently.
6. Page 10, line 289: Figure 5 is quoted, but Figure 4 did not appear before. It is recommended to check the continuity of chart numbers.
7. Page 4, Table 1: The last two lines refer to the same document "[17]", and the duplicate lines should be deleted.
8 8. Page 18-21 References, some of which lack periodical names, issue numbers or page numbers (such as [1] and [16]), please complete them according to periodical requirements.
9. Page 5, the working principle of CRISPR/Cas9 is described repeatedly in sections 2.1 and 2.1.1 respectively, and it is suggested to merge and simplify.
10. page 11, line 337: "lee et al. used crispr/cass9 to introduce a38mutation ..." is the reference [54] correct? Please check whether it is the work of Koslova and others.
11. page 13, line 413: "plin 1 knockout cell morphology changed from spindle to elliptic" suggests supplementing cell types (such as chicken preadipocytes) to enhance readability.
12. The contents of "funding" and "Acknowledgments" are completely repeated, so it is suggested to delete one of them.
13. Abbreviations such as "LNP, PEI, PLGA" appear in the article, but they are not listed in the final Abbreviations list, so it is suggested to supplement them.
Author Response
Manuscript ID: vetsci-4291577
Title: Application of Gene Editing Technology in Poultry
Authors: Ruyu Liao, Rong Ran, Yixin Liu, Xinyi Zhou, Min Tan, Qigui Wang, Haiwei Wang, Xi Lan *
Dear Reviewers,
Thank you very much for your valuable comments and suggestions on our manuscript. We have carefully considered all the points raised by the reviewer and have revised the manuscript accordingly. Below we provide a point-by-point response to each comment. All modifications have been highlighted in the revised manuscript.
Comments 1: Reviewer's comment: "page 2, line 40: 'with traditional ge' is obviously truncated and should be completely replaced by 'with traditional gene editing technologies ...'
Response 1: Thank you for pointing out this typographical error. We have corrected this sentence on Page 2, Line 40. The truncated phrase "with traditional ge" has been replaced with "with traditional gene editing technologies".
Comments 2: Reviewer's comment: "Page 3, line 105: 'The crispr sequence includes a leader region ...' The sentence is slightly lengthy, and it is suggested to split it into short sentences."
Response 2: Thank you for this suggestion. We have split the lengthy sentence on Page 3, Line 105 into shorter sentences for better readability.
Comments 3: Reviewer's comment: "Page 6, line 145: 'Nick ase CAS9 (NCAS9) is generated by deactivating one of the nuclear domains ...' It is suggested to change it to 'is generated by activating one of the nuclear domains'."
Response 3: Thank you for this correction. We have changed "deactivating" to "activating" on Page 6, Line 145.
Comments 4: Reviewer's comment: "In this paper, 'gene editing' and 'genome editing' are mixed, and it is suggested to unify them into 'genome editing' or 'gene editing'."
Response 4: Thank you for this important suggestion. We have carefully reviewed the entire manuscript and unified the terminology. We have chosen to use "gene editing" as the primary term throughout the manuscript, as it is more commonly used in the context of agricultural animal applications. All instances of "genome editing" have been changed to "gene editing" except in direct quotations or references where the original term is preserved.
Comments 5: Reviewer's comment: "The names of diseases such as 'Avian Leukosis' and 'avian influenza' should be capitalized consistently."
Response 5: Thank you for this observation. We have standardized the capitalization of disease names throughout the manuscript. Disease names are now consistently capitalized (e.g., "Avian Leukosis", "Avian Influenza").
Comments 6: Reviewer's comment: "Page 10, line 289: Figure 5 is quoted, but Figure 4 did not appear before. It is recommended to check the continuity of chart numbers."
Response 6: Thank you for catching this numbering error. We have rechecked the figure numbering throughout the manuscript and have revised it accordingly to ensure continuity.
Comments 7: Reviewer's comment: "Page 4, Table 1: The last two lines refer to the same document '[17]', and the duplicate lines should be deleted."
Response 7: Thank you for identifying this redundancy. We have reviewed Table 1 and the duplicate entry has now been removed.
Comments 8: Reviewer's comment: "Page 18-21 References, some of which lack periodical names, issue numbers or page numbers (such as [1] and [16]), please complete them according to periodical requirements."
Response 8: Thank you for this careful check. We have thoroughly reviewed all references and completed the missing information for each entry. As the modifications are extensive, they are not listed here one by one; the specific revisions have been made in the main text.
Comments 9: Reviewer's comment: "Page 5, the working principle of CRISPR/Cas9 is described repeatedly in sections 2.1 and 2.1.1 respectively, and it is suggested to merge and simplify."
Response 9: Thank you for this constructive suggestion. We have merged and simplified the description of the CRISPR/Cas9 working principle, and the revisions have been made in the main text.
Comments 10: Reviewer's comment: "page 11, line 337: 'lee et al. used crispr/cass9 to introduce a38mutation ...' is the reference [54] correct? Please check whether it is the work of Koslova and others."
Response 10: Thank you for verifying this citation. We have carefully checked the reference on Page 11, Line 337. The original citation [54] was indeed incorrect. We have now corrected it, and the revision has been made in the main text.
Comments 11: Reviewer's comment: "page 13, line 413: 'plin 1 knockout cell morphology changed from spindle to elliptic' suggests supplementing cell types (such as chicken preadipocytes) to enhance readability."
Response 11: Thank you for this suggestion. We have supplemented the cell type on Page 13, Line 413, and the revision has been made in the main text.
Comments 12: Reviewer's comment: "The contents of 'funding' and 'Acknowledgments' are completely repeated, so it is suggested to delete one of them."
Response 12: Thank you for pointing out this duplication. We have checked the "Funding" and "Acknowledgments" sections. The duplication has been removed, and the revision has been made in the main text.
Comments 13: Reviewer's comment: "Abbreviations such as 'LNP, PEI, PLGA' appear in the article, but they are not listed in the final Abbreviations list, so it is suggested to supplement them."
Response13: Thank you for this careful observation. We have carefully checked all abbreviations throughout the manuscript and included them in the Abbreviations list. The revisions have been made in the main text. Additionally, the following abbreviations have been supplemented:
|
AAV |
Adeno-Associated Virus |
|
ALV-J |
Avian Leukosis Virus Subgroup J |
|
NDV |
Newcastle Disease Virus |
|
PGC |
Primordial Germ Cell |
|
SCNT |
Somatic Cell Nuclear Transfer |
|
SERS |
Surface-Enhanced Raman Scattering |
|
Rox |
6-Carboxy-X-Rhodamine |
|
PS |
Phosphate-Buffered Saline |
|
GGP |
Grandparent Generation Poultry |
|
GP |
Germline Progenito |
We appreciate the reviewer's thorough and constructive feedback, which has significantly improved the quality of our manuscript. We believe that all concerns have been adequately addressed and hope that the revised manuscript is now suitable for publication in Veterinary
Best regards,
Ruyu Liao
Xi Lan and Haiwei Wang (Corresponding Author)
College of Animal Science and Technology, Southwest University
Chongqing, China
Email: xlan104@swu.edu.cn
Tel: +86 15310871004
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis is a comprehensive review that outlines the development of gene editing technologies, discusses the advantages and limitations of key gene editing tools, and explores their applications in animals. The review examines the challenges and prospects of gene editing in animal husbandry, including off-target effects, ethical concerns, and technical complexities.
The objectives should be revised to include animals in general.
Abstract: good
Define all abbreviations for the first time
The introduction is well written; more citations are required.
In general, this is a very informative review, well-written and organized. More citations are needed in the text.
A subtitle of the applications of these methods on commercial strains of poultry is needed. The question is, could this technology be applied to commercial strains of poultry or just pure breeds? If this is the case, what are the implications for commercial strains?
For the key studies that were cited, please provide the breed (strain) of poultry.
Author Response
Manuscript ID: vetsci-4291577
Title: Application of Gene Editing Technology in Poultry
Authors: Ruyu Liao, Rong Ran, Yixin Liu, Xinyi Zhou, Min Tan, Qigui Wang, Haiwei Wang* and Xi Lan*
Dear Reviewers,
Thank you very much for your valuable comments and suggestions on our manuscript. We have carefully considered all the points raised by the reviewer and have revised the manuscript accordingly. Below we provide a point-by-point response to each comment. All modifications have been highlighted in the revised manuscript.
Comments 1: The objectives should be revised to include animals in general.
Response 1: Thank you for this constructive suggestion. We have revised the objectives section to expand the scope to cover animals in general while retaining the focus on poultry as the core application object, making the positioning of this review more comprehensive and accurate.
Comments 2: Abstract: good.
Response 2: We sincerely appreciate the reviewer’s positive comment on the abstract.
Comments 3: Define all abbreviations for the first time.
Response 3: Thank you for pointing out this issue. We have carefully checked the full text and supplemented the definition of all abbreviations at their first appearance, and unified the format to improve the readability and standardization of the manuscript.
Comments 4: The introduction is well written; more citations are required.
Response 4: We are grateful for the positive feedback on the introduction. According to the suggestion, we have added a number of classic and latest high-quality citations in the introduction section to strengthen the theoretical foundation and background narration.
Comments 5: In general, this is a very informative review, well-written and organized. More citations are needed in the text.
Response 5: Thank you very much for your positive and encouraging comments. We have further supplemented and optimized the citations in the main text, especially in the parts of technology development, application cases and prospect analysis, so that the literature support is more sufficient and rigorous.
Comments 6: A subtitle of the applications of these methods on commercial strains of poultry is needed. The question is, could this technology be applied to commercial strains of poultry or just pure breeds? If this is the case, what are the implications for commercial strains?
Response 6: Thank you for this key professional suggestion. We have added a new subtitle section focusing on the application of gene editing in commercial poultry strains. In this section, we clearly explain that gene editing can be applied to both pure lines and commercial crossbred strains; and systematically elaborate the application paths, technical advantages, breeding value and industrial implications in commercial poultry strains.
Comments 7: For the key studies that were cited, please provide the breed (strain) of poultry.
Response 7: Thank you for this rigorous comment. We have rechecked all key cited studies, and supplemented the corresponding poultry breed/strain information in the text one by one to make the description of research materials more accurate and standardized.
We appreciate the reviewer's thorough and constructive feedback, which has significantly improved the quality of our manuscript. We believe that all concerns have been adequately addressed and hope that the revised manuscript is now suitable for publication in Veterinary Sciences.
Best regards,
Ruyu Liao
Xi Lan and Haiwei Wang (Corresponding Author)
College of Animal Science and Technology, Southwest University
Chongqing, China
Email: xlan104@swu.edu.cn
Tel: +86 15310871004
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn the present research article entitled " Application of Gene Editing Technology in Poultry, , the authors explore Gene Editing Technology. Authors should improve the manuscript accordingly. Major comments are as follows:
- Table 1. Significant milestones in the advancement of gene-editing technology should be revised to reflect its application in poultry and to incorporate additional citations from the relevant research literature.
- The author should be included at the end of the review articles, along with the limitations of this technology, suggestions for future work, and any relevant clinical trial data.
- Applications of gene editing in poultry should be summarized in a table in section 3, making it clear and related to the application.
- Gene Editing Technology is an advanced technology, so the author should cite applications of this technology from 2021 to 2025. Most citations are from before 2021.
- Author should revise the Introduction to Gendered Terminology in Gene Editing Technology: A Deeper Exploration of Polarity.
- The author should explain Figure 1: The CRISPR/Cas9 gene editing process for livestock and poultry in the legends, like a step-by-step process
- Author should add one more table, a success story of Major milestones in the development of gene editing technology in the livestock field.
- Author should explain Figure 2 in the legends.
- Author should explain Figure 3. Structural diagrams of dCas9, nCas9, Cas12a and Cas13a.
- Author should be Salmonella (especially Salmonella typhimurium) line no 449 should be italic, and the rest of the names
The English language needs to improve
Author Response
Manuscript ID: vetsci-4291577
Title: Application of Gene Editing Technology in Poultry
Authors: Ruyu Liao, Rong Ran, Yixin Liu, Xinyi Zhou, Min Tan, Qigui Wang, Haiwei Wang, Xi Lan *
Dear Editor and Reviewers,
Thank you very much for your detailed and constructive comments on our manuscript. We have carefully considered all the points raised by the reviewer and have revised the manuscript accordingly. Below we provide a point-by-point response to each comment.
Comments 1：Table 1. Significant milestones in the advancement of gene-editing technology should be revised to reflect its application in poultry and to incorporate additional citations from the relevant research literature."
Response 1: Thank you for this important suggestion. We have revised Table 1 to focus specifically on milestones related to gene editing applications in poultry, rather than general gene editing technology development. We have also added additional citations from the relevant poultry research literature. The revised Table 1 now includes the following key milestones:
|
Year |
Technology |
Achievement |
|
2016 |
CRISPR-mediated editing in chicken primordial germ cells (PGCs) |
Efficient gene editing in chicken PGCs enabled stable germline transmission and opened a new era for avian transgenic research |
|
2016 |
Generation of gene-edited chickens |
CRISPR/Cas9 technology was successfully used to generate genetically modified chickens, demonstrating its feasibility in poultry breeding |
|
2020 |
Antiviral gene editing in poultry |
Genome editing targeting host antiviral genes emerged as a promising strategy for improving poultry disease resistance |
|
2020 |
ALV-J-resistant chickens via NHE1 editing |
Precise editing of the chicken NHE1 receptor conferred resistance to ALV-J infection, demonstrating the practical potential of CRISPR-assisted antiviral breeding in poultry |
Comments 2: Reviewer's comment: "The author should be included at the end of the review articles, along with the limitations of this technology, suggestions for future work, and any relevant clinical trial data."
Response 2: Thank you for this suggestion. We have expanded Section 4 to include the following details:
Limitations of the technology: Off-target effects, technical complexity, delivery challenges, and HDR inefficiency in non-dividing cells.
Suggestions for future work: Development of more precise editing tools (base editors, prime editors), improved delivery systems (nanoparticles, CPPs), AI-assisted gRNA design, and multi-omics integration for safety assessment.
Relevant clinical trial data: We have added a discussion of clinical trials, including the first CRISPR/Cas9 clinical trial for sickle cell disease and beta-thalassemia [20], as well as the ethical implications of human germline editing [88,89].
Comments 3: Reviewer's comment: "Applications of gene editing in poultry should be summarized in a table in section 3, making it clear and related to the application."
Response 3: Thank you for this suggestion. We have added Table 4 in Section 3, which summarizes the applications of gene editing technology in poultry organized by application area, target, representative genes, key methods, and achievements.
Comments 4: Reviewer's comment: "Gene Editing Technology is an advanced technology, so the author should cite applications of this technology from 2021 to 2025. Most citations are from before 2021."
Response 4: Thank you for this important observation. We have added numerous citations from 2021-2025 throughout the manuscript, and the specific revisions have been presented in the main text.
Comments 5: Reviewer's comment: "Author should revise the Introduction to Gendered Terminology in Gene Editing Technology: A Deeper Exploration of Polarity."
Response 5: Thank you for this insightful suggestion. We have revised the Introduction to include a discussion of "polarity" in gene editing technology. Specifically, we have added content explaining the functional polarity of CRISPR/Cas9 systems. Crucially, the DNA targeting and hybridization mechanism of the CRISPR/Cas9 sys-tem exhibits distinct structural and functional polarity. The recognition process is highly directional; following PAM recognition at the 3' end of the target DNA strand, the unwinding of the DNA double helix and the subsequent RNA-DNA heteroduplex formation (R-loop) propagate in a polar, zipper-like manner. This propagation pro-ceeds strictly from the PAM-proximal "seed" region (typically the 8–12 nucleotides ad-jacent to the PAM) towards the PAM-distal 5' end. This functional polarity fundamen-tally dictates the mismatch tolerance of the system: the PAM-proximal seed region is highly stringent and sensitive to base mismatches, whereas the PAM-distal end exhib-its significantly greater flexibility. Understanding this directional polarity is essential for optimizing gRNA design to maximize on-target cleavage efficiency while mitigat-ing off-target hybridization.
Comments 6: Reviewer's comment: *"The author should explain Figure 1: The CRISPR/Cas9 gene editing process for livestock and poultry in the legends, like a step-by-step process."
Response 6: Thank you for this suggestion. We have revised the legend of Figure 1 to provide a detailed, step-by-step explanation of the CRISPR/Cas9 gene editing process for both livestock and poultry.
Revised Figure 1 legend: "Figure 1. The CRISPR/Cas9 gene editing process for livestock and poultry. Step 1: Design of specific sgRNAs targeting the gene of interest and construction of CRISPR/Cas9 expression vectors. Step 2: Delivery of editing components into target cells. For poultry, primordial germ cells (PGCs) are extracted and transfected in vitro. For livestock, somatic cells are edited and utilized as donor cells. Step 3: Generation of founder animals. Edited PGCs are injected into developing chicken embryos to produce chimeric founders (F0). For livestock, reconstituted zygotes from somatic cell nuclear transfer (SCNT) are transplanted into surrogate mothers. Step 4: Validation of heritable edits through genotypic PCR, genomic sequencing, and off-target analysis. Step 5: Sequential generational screening to establish homozygous edited lineages with stable inheritance of targeted traits."
Comments 7: Reviewer's comment: "Author should add one more table, a success story of Major milestones in the development of gene editing technology in the livestock field."
Response 7: Thank you for this suggestion. We have added Table 3 presenting a timeline of major milestones in the development of gene editing technology for livestock (including sheep, pigs, cattle, goats, and chickens). The table covers the period from 1997 (Dolly the sheep) to 2025 (multi-omics integration), highlighting key success stories such as MSTN knockout pigs, POLLED gene editing in cattle, CD163 knockout for PRRSV resistance, BLG knockout for hypoallergenic milk, and KISS1 knockout for castration-free trait.
Comments 8: Reviewer's comment: "Author should explain Figure 2 in the legends."
Response 8: Thank you for this suggestion. We have revised the legend of Figure 2 to provide a clear explanation of the structural differences between ZFN, TALEN, and CRISPR/Cas9 systems.
Revised Figure 2 legend: "Figure 2. Schematic diagram of the structure of ZFN, TALEN and CRISPR/Cas9. (A) Zinc Finger Nucleases (ZFNs) consist of customizable zinc-finger DNA-binding proteins (each recognizing 3-4 nucleotides) fused with a FokI endonuclease cleavage domain. Dimerization of FokI is required for DNA cleavage. (B) Transcription Activator-Like Effector Nucleases (TALENs) utilize TALE repeat arrays (each repeat recognizing a single nucleotide) for DNA recognition, similarly tethered to a FokI nuclease. (C) The CRISPR/Cas9 system employs a single guide RNA (sgRNA) to direct the Cas9 nuclease to a specific genomic locus adjacent to a Protospacer Adjacent Motif (PAM). The Cas9 enzyme utilizes its HNH and RuvC domains to introduce a double-strand break (DSB) 3-4 nucleotides upstream of the PAM. The resulting DSBs are subsequently repaired either by Non-Homologous End Joining (NHEJ) or by Homology-Directed Repair (HDR) in the presence of a repair template."
Comment 9: Reviewer's comment: "Author should explain Figure 3. Structural diagrams of dCas9, nCas9, Cas12a and Cas13a."
Response 9: Thank you for this suggestion. We have revised the legend of Figure 3 to provide a detailed explanation of each Cas variant.
Revised Figure 3 legend: "Figure 3. Structural diagrams of dCas9, nCas9, Cas12a and Cas13a. (A) Dead Cas9 (dCas9) contains inactivating mutations in both the RuvC and HNH nuclease domains (typically D10A and H840A), allowing it to bind to target DNA without causing cleavage. This property is utilized for gene regulation (CRISPRa/CRISPRi) by fusing dCas9 with transcriptional activators or repressors. (B) Nickase Cas9 (nCas9) possesses a mutation in one of the nuclease domains (either RuvC or HNH), generating single-stranded DNA nicks rather than double-strand breaks. When paired with dual sgRNAs targeting opposite strands, this 'double-nicking' strategy reduces off-target effects by 50-1500 fold compared to wild-type Cas9. (C) Cas12a (Cpf1) relies on a single crRNA (no tracrRNA required) and recognizes T-rich PAM sequences (5'-TTTN-3'), creating staggered double-strand breaks distal to the PAM (18-23 nucleotides downstream). (D) Cas13a specifically targets and cleaves single-stranded RNA (ssRNA) and possesses a unique collateral trans-cleavage activity upon target recognition, which cleaves nearby non-target RNAs. This property is widely utilized in diagnostic biosensors for nucleic acid detection."
Comments 10: Reviewer's comment: "Author should be Salmonella (especially Salmonella typhimurium) line no 449 should be italic, and the rest of the names."
Response 10: Thank you for pointing out this formatting error. We have corrected the italicization of species names throughout the manuscript.
We greatly appreciate the reviewer's thorough and constructive comments, which have significantly improved the quality and completeness of our manuscript. We believe that all concerns have been adequately addressed and hope that the revised manuscript is now suitable for publication in Veterinary Sciences.
Best regards,
Ruyu Liao
Xi Lan and Haiwei Wang (Corresponding Author)
College of Animal Science and Technology, Southwest University
Chongqing, China
Email: xlan104@swu.edu.cn
Tel: +86 15310871004
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsAccpeted
Comments on the Quality of English LanguageThe English language needs to improve
