Base Editing of EUI1 Improves the Elongation of the Uppermost Internode in Two-Line Male Sterile Rice Lines

Round 1

Reviewer 1 Report

The manuscript by Dr. Tang and colleagues reported the generation of precise A-to-G editing in a rice male sterile line by using CRISPR-BE. The study was nicely executed and represented in this manuscript. However, the manuscript is needed to be improved on several sections, I have some suggestions.

 

1.     It would be helpful for the authors to discuss the rationale behind the study, including the motivation for studying base editing in a rice male sterile line and the potential applications of the research.

 

2.     Conducting next generation sequencing of the target amplicon could provide further information on the accuracy of the base editing process.

 

3.     It would be useful for the authors to consider the potential off-target activity of the ABE and to perform targeted amplicon deep sequencing to assess any unintended effects.

 

4.     It is not clear whether the authors tried using "biolistic delivery" of the editor. It would be helpful for the authors to include this information in the manuscript.

 

5.     The experimental and results section regarding the GA bioactivity assay could be improved by providing more detailed explanations and clarifying any unclear points.

 

6.     The figure 1E was not clear and it would be helpful for the authors to indicate the mutation more clearly in the models. I am wondering how a point mutation change the overall structure of the protein that they were not aligning?  It would also be useful for the authors to discuss how the point mutation affects the overall structure of the protein and the disruption of the protein's internal interactions. Removing figure 1D to the supplementary section may also be helpful.

Author Response

Dear reviewer,

     Thank you very much for your comments and professional advice. These opinions help to improve academic rigor of our article. Based on your suggestion and request, we have made corrected modifications on the revised manuscrpt. Meanwhile, the manuscript had be reviewed and edited by language services of MDPI. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Point 1: It would be helpful for the authors to discuss the rationale behind the study, including the motivation for studying base editing in a rice male sterile line and the potential applications of the research.

Response 1: Thank you for your suggestions. Knockout of EUI1 using the CRISPR/CAS9 tool often results in excessive elongation of the uppermost internode (Supplementary Figure 2). Since ABE only replaces the base of the target sequence, it will not destroy the integrity of the gene. Therefore, our research idea is to inhibit the oxidation activity of EUI1 to GA through ABE, so as to appropriately increase the content of GA in the uppermost internode, thereby improving its heading ability, reducing lodging risk, and improving the yield of hybrid seed production.

Point 2: Conducting next generation sequencing of the target amplicon could provide further information on the accuracy of the base editing process.

Response 2: Thank you for your suggestions. Figure 1. C is the peak map of our detection of mutant targets in transgenic plants. I have provided you with the complete sequencing results, please check in the attachment.

Point 3:  It would be useful for the authors to consider the potential off-target activity of the ABE and to perform targeted amplicon deep sequencing to assess any unintended effects.

Response 3: Thank you for your suggestions. About the ABE gene editing technology we used was different from CAS9, so no off-target verification experiments were conducted, but the phenotype of the plants was as expected.

Point 4: It is not clear whether the authors tried using "biolistic delivery" of the editor. It would be helpful for the authors to include this information in the manuscript.

Response 4: Thank you for your suggestions. ABE is a base editor based on CAS9, but this ABE base editor does not need PAM sequences and has a low miss rate, and can make base substitution for target sequences more accurately.

Point 5: The experimental and results section regarding the GA bioactivity assay could be improved by providing more detailed explanations and clarifying any unclear points.

Response 5: Thank you for your suggestions. Regarding the details of GA hormone content detection, we have provided the detection report of the testing company in the attachment, please check.

Point 6: The figure 1E was not clear and it would be helpful for the authors to indicate the mutation more clearly in the models. I am wondering how a point mutation change the overall structure of the protein that they were not aligning?  It would also be useful for the authors to discuss how the point mutation affects the overall structure of the protein and the disruption of the protein's internal interactions. Removing figure 1D from the supplementary section may also be helpful.

Response 6: Thanks for your suggestion, we have changed figure 1D into attached Supplementary Figure 2.

Author Response File: Author Response.docx

Reviewer 2 Report

The reviewer comments on the manuscript entitled “Base editing of EU11 improves the elongation of the uppermost internode in two-line male sterile rice line”. In this study, base editing was used to edit EUI1 to inhibit the epoxidation of gibberellin by EUI1. The increased content of active gibberellin in transgenic plants promoted the elongation of the uppermost internode of MSLs, which increased the panicle exsertion rate and outcrossing seed setting rate. The results is useful for understanding the molecular mechanisms of EUI1 regulation on uppermost internode elongation and improving the efficiency of hybrid seed production. After reviewing this manuscript, minor revision need to be addressed in this manuscript in order to be suitable for publication. 

1, Page 1, line 7,need change “ that plays a key role in panicle exsertion from the flag leaf sheath in rice (Oryza sativa) into “ that plays a key role in panicle exsertion from the flag leaf sheath in rice (Oryza sativa L.)”

2, Page 1, line 13, need change “the wild type (0.70ng/g, 0.57ng/g, and 0.42ng/g)” into “the wild type (0.70, 0.57, and 0.42ng/g)”.

3, Page 9, line 200-201, need change “EUI1 inactivates GA4 GA9 and GA12 through epoxidation (Figure 4A)” into “EUI1 inactivates GA4, GA9, and GA12 through epoxidation (Figure 4A)”.

4, Page 11, line 231-232, need change “B, C815S, eui1-1, and eui1-2 Seed setting rate , C,  C815S, eui1-1, and eui1-2 panicle wrapping rate.” into “B, Seed setting rate of C815S, eui1-1, and eui1-2. C, Panicle wrapping rate of C815S, eui1-1, and eui1-2.

5, Page 11, line 235,need change “3.6 Analysis of YGL53 in 3K” into “3.6 Analysis of EUI1 in 3K”.

 

Author Response

Dear reviewer,

     Thank you very much for your comments and professional advice. These opinions help to improve academic rigor of our article. Based on your suggestion and request, we have made corrected modifications on the revised manuscrpt. Meanwhile, the manuscript had be reviewed and edited by language services of MDPI. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Point 1: Page 1, line 7,need change “ that plays a key role in panicle exsertion from the flag leaf sheath in rice (Oryza sativa) into “ that plays a key role in panicle exsertion from the flag leaf sheath in rice (Oryza sativa L.)”

Response 1: Thank you for your suggestion, we have modified as required.

Point 2: Page 1, line 13, need change “the wild type (0.70ng/g, 0.57ng/g, and 0.42ng/g)” into “the wild type (0.70, 0.57, and 0.42ng/g)”.

Response 2: Thank you for your suggestion, we have modified as required.

Point 3: Page 9, line 200-201, need change “EUI1 inactivates GA4 GA9 and GA12 through epoxidation (Figure 4A)” into “EUI1 inactivates GA4, GA9, and GA12 through epoxidation (Figure 4A)”.

Response 3: Thank you for your suggestion, we have modified as required.

Point 4: Page 11, line 231-232, need change “B, C815S, eui1-1, and eui1-2 Seed setting rate , C,  C815S, eui1-1, and eui1-2 panicle wrapping rate.” into “B, Seed setting rate of C815S, eui1-1, and eui1-2. C, Panicle wrapping rate of C815S, eui1-1, and eui1-2.

Response 4: Thank you for your suggestion, we have modified as required.

Point 5: Page 11, line 235,need change “3.6 Analysis of YGL53 in 3K” into “3.6 Analysis of EUI1 in 3K”.

Response 5: Thank you for your suggestion, we have modified as required.

Author Response File: Author Response.docx

Reviewer 3 Report

Study entitled “Base editing of EU11 improves the elongation of the uppermost internode in two-line male sterile rice line” contains novel information which can further strengthen the existing knowledge of the field. Scientists planned their study according to need of time. Please consider addressing following concerns and find the attachment to improve the draft quality. The raised concerns may be incorporated before any consideration to publish this work.

1. Arrange your introduction section in systematic scientific way and identify the study gap and provide a robust hypothesis. Try to avoid stating general information and be specific.
2. The introduction is directionless without synthesis of literature, hypothesis or study question. It may be improved accordingly.
3. Improve the reporting language and avoid jargon. Make the results section concise and specific.
4. Some typo mistakes are observed while reviewing the draft. Authors are requested to read it very carefully and improved the draft accordingly.
5. Use journal’s guidelines for the format of references within text and at the end.
6. English language improvement looks necessary throughout the manuscript.

For further details, find the attachment please.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

     Thank you very much for your comments and professional advice. These opinions help to improve academic rigor of our article. Based on your suggestion and request, we have made corrected modifications on the revised manuscrpt. Meanwhile, the manuscript had be reviewed and edited by language services of MDPI. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Point 1: Arrange your introduction section in systematic scientific way and identify the study gap and provide a robust hypothesis. Try to avoid stating general information and be specific.

Response 1: Thank you for your suggestion. We have made modifications to the problem you raised.

Point 2: The introduction is directionless without synthesis of literature, hypothesis or study question. It may be improved accordingly.

Response 2: Thank you for your suggestion. We have made modifications to the problem you raised.

 

Point 3: Improve the reporting language and avoid jargon. Make the results section concise and specific.

Response 3: Thank you for your suggestion. For the problem you raised, we have found a professional paper revision agency to revise it. Please check the attachment for proof of modification.

Point 4: Some typo mistakes are observed while reviewing the draft. Authors are requested to read it very carefully and improved the draft accordingly.

Response 4: Thank you for your suggestion. We have made modifications to the problem you raised.

Point 5: Use journal’s guidelines for the format of references within text and at the end.

Response 5: Thank you for your suggestion. We have made modifications to the problem you raised.

Point 5: English language improvement looks necessary throughout the manuscript.

Response 6: Thank you for your suggestion. We regret there were problems with the English. The paper has been carefully revised by a professional language editing service to improve the grammar and readability. For further details, find the attachment please.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors, thank you for providing the explanations against each comments. I am sorry to say but, I think you still need to do the additional experiments related to next generation sequencing and off-target editing as its a separate editing experiments. Each base editing experiment need to be carefully monitored for potential off-target edits. The manuscript has the potential to be published but after the improvisation. 

Author Response

We have supplemented the off-target experiment of EUI1。According to the miss rate forecasting tool select the three most likely off-target gene (http://skl.scau.edu.cn/offtarget/) and the off-target rate detection for T₁ generation of mutant; The results showed that the 30 mutant strains did not have the mutation of the other three genes.

Author Response File: Author Response.docx