Construction and Functional Evaluation of CRISPR/Cas9 Multiple Knockout Vectors of the FAD2 Gene Family
Round 1
Reviewer 1 Report (Previous Reviewer 1)
Please find the comments at the attachment. I didn't find the answers for my previous comments and corrections. I didn't find the Supplementary material.
Comments for author File: 
Comments.pdf
Please find the comments at the attachment. This is not the revised version. I didn't find the answers for my previous comments and corrections. I didn't find the Supplementary material.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report (New Reviewer)
Zhang et al., in the manuscript entitled” Construction and functional evaluation of CRISPR/Cas9 multiple knockout vectors for the FAD2 gene family” evaluated function of FAD2 gene family in soybean by using CRISPR/Cas9 gene editing procedure. Although the article has positive points in terms of the methods used and the results obtained, it still suffers from some significant shortcomings in terms of materials and statistical methods used for data analysis. Therefore, before the possible publication of this article, the authors are advised to pay attention to the following points and improve them.
General comments
The study goals are not addressed in the introduction section, so the last section of your introduction and point out your main goals of your study.
In the material and method section it is not explained thoroughly the Agrobacterium-mediated method of transformation. Agrobacterium tumefaciens or Agrobacterium rhizogenes? However, only in results section it is found that Agrobacterium tumefaciens is used, but authors are advised to clarify this issue in the material and method section. In the other hand, the Agrobacterium tumefaciens mediated transformation o soybean is a critical step and authors should explain the transformation procedure by referring to relevant previous studies.
Specific comments
Line 46: When a word appears for a first time in the text (e.g., FAD2) writ out the full phrase then use its abbreviation.
Line 48: if FAD2 used as an enzyme name it should be not in italic format.
Line 54: Camelina sativa is a scientific name and should be written in italic format.
Line 69: references?
Line 75: “period,” use dot instead of virgule
Line 75: “these three strains” which strains?
Line 279: “on September 20,000 )” ?? check this part.
Line 95: “Figures 2” how about the figure 1?
Line 112: Since the method is not explained in the material and method section the results for the transformation section is not satisfying and need more details. For example C and D section of the figure 3 is not addressed in the text.
Line 122: The percentage of positive transformed soybean strains are not shown in the results!
Line 125: the caption for the figure 4 is too summarized and need more details.
Line 128: point out the chamber condition for soybean growing.
Line 170: “q-RTPCR” use RT-qPCR instead of q-RTPCR
Line 175: the figure quality is too low.
Line 178: improve the end part of the caption by using appropriate words like “respectively”.
Line 195 and 197: why authors did not anlysed the TAGs content in the soybean mutant lines, since these are principal components of soybean seed oil.
Line 199: LSD or Duncan? p = 0.05. or P ≤ 0.05? The statistical analyzes (in Results and materials and methods sections) are incomplete in terms of explanations and use of symptoms, and there is no unity of procedure in them, and they should be fundamentally revised in different parts of the article.
Line 287: “Escherichia coli” is a scientific name and should be written in italic format.
Line 294-295:
Line 332: Which method (GC or GC-MS) was used to detect the fatty acids? If only GC method has used, so how did you detect the compounds?
Line 337: The extraction procedure and used instruments (e.g., Soxhlet or rotary evaporator) is not shown.
Line 354-355: improve the sentence.
Line 360: “ANOVA Duncan text.”?? Improve this section.
Line 361: “P” should be in italic format.
Conclusion section or a paragraph addressing the main findings of the study is not determined in the manuscript.
Author Response
Please see in the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report (Previous Reviewer 1)
Please find the comments in the attachment.
Comments for author File: Comments.pdf
Author Response
"Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report (New Reviewer)
-check the scientific names format (line 294 and 296)
- line 359-360 non-significant or significant?
Author Response
Please see in the attachment.
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Kindly find the comments at the attachment.
Comments for author File: 
Comments.docx
Reviewer 2 Report
The paper reports the results of a genome editing approach of soybean FAD2 genes to modify the oil content and omega 3/omega 6 ratio in soybean seeds. The work is not particularly original, and the experimental approach is very poorly described.
The editing approach is poorly described and above all the steps for construct preparation are not explained in detail (which vectors? how were the gRNAs cloned? etc.). It is not clearly explained how the different amplifications were done for the verification of the transgenes as well as for the amplification of the target regions. The positions of the sgRNAs in the corresponding target genes are not indicated. The effects of the editing on the target proteins are not indicated, and for this reason it is not clear how the low levels of FAD2 genes expression can be explained: ex. low mRNA expression or absence of mRNAs due to early interruptions in synthesis?
In general, the work needs to be deeply revised in terms of the English language, presentation of the methods and results and obviously also the discussion
