CRISPR/Cas9-Mediated Genome Editing of Soluble Starch Synthesis Enzyme in Rice for Low Glycemic Index

Round 1

Reviewer 1 Report

The manuscript titled "CRISPR/Cas9-mediated Genome Editing of Rice for Low Glycemic Index" involves genome editing of soluble starch synthase enzyme using the CRISPR-Cas9 system to generate rice cultivars with low glycemic index. The development of such cultivars is essential in the current scenario with increased incidence rates of type II diabetes. 

However, the presentation of scientific images in this manuscript does not follow publication criteria. The labeling arrows for DNA gel images come out from all directions. In principle, labeling is done only in the left direction with clear description on top about lanes. All gels have an unclear labeling M, M1 and M6 which is highly misleading. There is no description of the ladder (company details). 

Details about the guide RNA sequence and how the guide RNA was selected are missing. Was there any particular software used to search for guide RNA sequence? 

In many places, one cannot see any space for example, in Fig 2- "Theformation." Even the abbreviations used in the references are incorrect at multiple locations. 

Figures related to cloning can be moved to the supplementary and "Result" sections can directly start from plant transformation with a little description on cloning strategy. Labeling of gel images should only be towards the left side of the gel. For example, in Figure 4, one can see arrows coming from the middle, left, and right of the gel. 

In some places, the author says biallelic (Line 23)and in some, they say diallelic (Line 65). Similarly, there are many other inconsistencies. 

Author Response

Reviewer 1

1) The manuscript titled "CRISPR/Cas9-mediated Genome Editing of Rice for Low Glycemic Index" involves genome editing of soluble starch synthase enzyme using the CRISPR-Cas9 system to generate rice cultivars with low glycemic index. The development of such cultivars is essential in the current scenario with increased incidence rates of type II diabetes.

Response: Thanks

2) However, the presentation of scientific images in this manuscript does not follow publication criteria. The labeling arrows for DNA gel images come out from all directions. In principle, labeling is done only in the left direction with clear description on top about lanes. All gels have an unclear labeling M, M1 and M6 which is highly misleading. There is no description of the ladder (company details).

Response: All figures have been corrected as per the suggestion of the reviewer.

3) Details about the guide RNA sequence and how the guide RNA was selected are missing. Was there any particular software used to search for guide RNA sequence?

Response: The relevant information has been updated in the manuscript.

4) In many places, one cannot see any space for example, in Fig 2"Theformation." Even the abbreviations used in the references are incorrect at multiple locations.

Response: Required corrections have been made.

5) Figures related to cloning can be moved to the supplementary and "Result" sections can directly start from plant transformation with a little description on cloning strategy. Labeling of gel images should only be towards the left side of the gel. For example, in Figure 4, one can see arrows coming from the middle, left, and right of the gel.

Response: The needful has been done.

6) In some places, the author says biallelic (Line 23) and in some, they say diallelic (Line 65). Similarly, there are many other inconsistencies.

Response: The same has been corrected

Reviewer 2 Report

I have gone through the manuscript “CRISPR/Cas9-mediated genome editing of rice for low glycemic index”. The work done by the authors is of great concern in developing low GI rice by employing the CRISPR-Cas9 genome editing tool. Successful development of knockout rice lines of the starch synthase genes to create high-amylose rice is something of doing worth. Before the manuscript be accepted for publication, the authors should address the following concern:

Comments:

Title: Please see if the name of the gene targeted in the rice can be mentioned.

Abstract: Please use ‘articles’ appropriately; for example, add ‘the’ before Mendelian. The abstract could be further revised in terms of scientific and grammatical quality.

Introduction: This section is well written, however, needs a revision for proper English. Line 37, 40, 60, etc. needs attention. Also, give breaks to create more than one sentence.

Materials and Methods: Methods are well explained. Verification of the sequence of studied parameters should be in agreement with the sequence of results. Some fragments of M&M are seen in the ‘Results’ section. Please place them only in the M&M. Why there is no mention of any Statistical procedures?

Results: This section is starting with a feeling of ‘Materials & Methods’ and hence needs a proper framing of the ‘Results’ only. Shift sentences of M&M to the relevant section. However, the depiction of results is good, and ample data has been generated and presented. Figures are of good quality with self-explanatory legends. Again, long sentences and many typos. Please revise. The tables are also good. The presentation of data needs better clarity, please do that. Most of the figures are of high quality and presented in impressive ways such as in Figure 3 and Figure 11. There is still a margin to improve the presentation of other Figues in the manuscript. The targeted genes and the gRNAs used are properly mentioned. The methods claim that NGS is used for genotyping are acceptable and in results do not show the frequency of the different KO alleles produced for the different lines.

There are many tools for targeted mutagenesis available, engineered nucleases are easy, efficient and precise players which depend upon site specific recombinases and nuclease-free homologous recombination, and I cannot understand why homologous recombination is mentioned.

The discussion and conclusions are coherent with the results, clear and directly relevant to the research data.

References: Up to date and relevant. I can see most of the references are not properly formatted. Please revise the entire section.

Since the objectives of the present study are well identified, a proper methodology was adopted and the results are properly presented, therefore, I recommend the manuscript for acceptance once the above-raised issues are addressed.

Author Response

Reviewer 2

Title: Please see if the name of the gene targeted in the rice can be mentioned.

Response: The name of targeted gene has been updated in the manuscript.

Abstract: Please use ‘articles’ appropriately; for example, add ‘the’ before Mendelian. The abstract could be further revised in terms of scientific and grammatical quality.

Response: Entire manuscript has been revised for English grammar.

Introduction: This section is well written, however, needs a revision for proper English. Line 37, 40, 60, etc. needs attention. Also, give breaks to create more than one sentence.

Response: Thanks. All corrections have been made.

Materials and Methods: Methods are well explained. Verification of the sequence of studied parameters should be in agreement with the sequence of results. Some fragments of M&M are seen in the ‘Results’ section. Please place them only in the M&M. Why there is no mention of any Statistical procedures?

Response: Updated as per suggestions

Results: This section is starting with a feeling of ‘Materials & Methods’ and hence needs a proper framing of the ‘Results’ only. Shift sentences of M&M to the relevant section. However, the depiction of results is good, and ample data has been generated and presented. Figures are of good quality with selfexplanatory legends. Again, long sentences and many typos. Please revise. The tables are also good. The presentation of data needs better clarity, please do that. Most of the figures are of high quality and presented in impressive ways such as in Figure 3 and Figure 11 There is still a margin to improve the Figure 3 and Figure 11. There is still a margin to improve the presentation of other Figures in the manuscript. The targeted genes and the gRNAs used are properly mentioned. The methods claim that NGS is used for genotyping are acceptable and in results do not show the frequency of the different KO alleles produced for the different lines.

Response: Revised as suggested

 There are many tools for targeted mutagenesis available, engineered nucleases are easy, efficient and precise players which depend upon site specific recombinases and nucleasefree homologous recombination, and I cannot understand why homologous recombination is mentioned.

Response: Updated as per requested

 The discussion and conclusions are coherent with the results, clear and directly relevant to the research data.

Response: Thanks

References: Up to date and relevant. I can see most of the references are not properly formatted.

Response: The references have been revised for proper formatting