Research Trends and Challenges of Using CRISPR/Cas9 for Improving Rice Productivity

Round 1

Reviewer 1 Report

The manuscript contains interesting and relevant information. In my opinion, it could be significantly improved to provide good value to the reader.

My major concern is that it should be clear what the scope of the manuscript is. Having read the title and the abstract, I was a bit lost when reading the following chapters of the manuscript.

It should be made very clear which role the possible application of the CRISPR/Cas9-technology has in this context. At least this is what I would expect from reading the abstract as it proposes CRISPR/Cas9 to be an important technology contributing to solve the challenges for rice production. Also, the introduction points towards this aspect.

Personally, I would like to read more about the concrete challenges, and how the current and future trends in CRISPR/Cas9 research in rice can contribute.

I recommend to consider concise and easily comprehensible argumentation lines concerning the concrete challenges for rice production and potential solutions. Following a clear argumentation line throughout the manuscript would help the reader to understand the challenges (e.g. in the introduction), leading him/her to why research on certain traits is important and finally to the potential solutions as presented in the manuscript.

For instance, I had difficulties following the rationale in chapter 3.1., but I consider chapter 3.1. very important for the manuscript. Unfortunately, it is not very useful for the scope of the manuscript in its current form. In my opinion, it should be expanded and specifically explain the challenges of rice transformation and the application of genome editing in rice in more detail.

I very much like the idea of Figure 3, as it nicely explains the steps required to perform genome editing. However, it may be improved significantly; some remarks: Please check the captions in the pictures for spelling and accuracy of information; please rephrase the legend in order to fit the pictures precisely. Please reconsider the following points: caption of picture A, as “gene targeting” might not be the right wording; it is not clear where the rice callus for transformation comes from as this step is missing in the explanation and please rephrase the corresponding legend; picture F: please make the message clear to the reader; picture “I” might be misleading: was it your intention to describe a “super plant” that includes all the mentioned traits?

The following chapters contain interesting information and correspond to the scope of the manuscript. I would suggest to design an argumentation line and to balance the amount of the information better. As a reader, I would like to be convinced that the presented traits are indeed selected based on the importance in rice breeding, and it would be interesting to see challenges and success stories in a systematic and easily understandable way. For example, male sterility is important to facilitate rice breeding, which is the tool to solve the challenges in rice production by targeting specific traits. This should be presented in a clear way. Currently, I do not see a rationale behind the selection of papers.

The authors frequently switch between general and specific topics, which makes the text hard to follow for the reader.

The text contains redundancies, e.g. lines 46-47. I would also like to mention that Chapter 2 on the general mechanisms of the CRISPR/Cas9 system could be significantly shortened for the benefit of discussing the challenges in rice production in a more detailed way. It would be of value to also focus more on the specificities of using the system in plants. I furthermore recommend taking care of the relevance and accuracy of descriptions (e.g. lines 74 ff.).

I agree that it is important to point out the differences between NHEJ and HDR, as this is of major relevance when designing experiments targeting the improvement of plant characteristics. The figure the authors presented is of a general nature and has already been presented in various forms and details in a number of papers. For this reason, it might be redundant to state an original source; however, if the figure has been designed/modified based on a specified source it should be mentioned.

Chapter 4: This chapter should contain a discussion on the trends (e.g., can CRISPR/Cas9 target the right traits and thus indeed improve rice productivity?), and the challenges of CRISPR/Cas9 applications in rice breeding. I suggest to include a brief analysis of the trends displayed in the previous chapter and how useful they are to enhance rice breeding. Are there traits that have not been targeted so far and what may be the reasons. It would be interesting to read a discussion why CRISPR/Cas9 can make a major contribution and be a significant addition to traditional rice breeding. Like this, you would put Figure 4 into better context.

The idea of Figure 4 is appreciated. However, please check it for accuracy, including the legend!

Remarks on specific content:

Please also be very careful when stating “genome-edited crops do not carry transgenesis DNA…”. This sentence should be rephrased. Although frequently the case, it is not correct to state that genome edited crops are always free of foreign DNA.

Please consider that sentences like the ones in 249-252 do not seem to be in the right place.

I would like to see more attention in delivering the messages, to avoid readers spending time to figure out the exact meaning of messages: e.g. line 84: “The CRISPR sequence…” or line 131: “The most direct use…”, etc.

The following sentences are not accurate or misleading in their current wording: “traditional GMO regulations are circumvented” (lines 138-139), “Genome edited crops do not…” (lines 557-558) and “Transgenic organisms are strictly regulated…” (lines 559-562). These sentences should be reconsidered to provide correct information to the reader. Please also avoid sentences like: “CRISPR/Cas9 system has potential…” (line 142), as they do not deliver relevant information, or rephrase them. It is very relevant though to explain the advantages and the potential of the technology (as in lines 145 ff.).

Throughout the article, the authors do not clearly distinguish between breeding techniques, and tools and/or methods that are used for breeding. (e.g., lines 13 ff.)

Remarks on spelling and style:

Concerning spelling and English style, there are many missing definite/indefinite articles. In addition, there is a remarkable amount of very long sentences that are hard to comprehend.

The spelling and font type of gene, species, and mutant names (e.g., italic; use of capital and small letters;…) has to be carefully checked throughout the manuscript.

Spelling of CRISPR/Cas9 needs to be unified.

Is there something missing in the sentence “These findings…” (lines 298-299)?

Table 1. Check spelling and font types.

Author Response

Responses to Reviewer 1:

We are grateful for the reviewer on valuable comments and suggestions for the manuscript. We believe that the comments have identified important areas that required improvement. The revisions or answers are as follows:

The manuscript contains interesting and relevant information. In my opinion, it could be significantly improved to provide good value to the reader.

My major concern is that it should be clear what the scope of the manuscript is. Having read the title and the abstract, I was a bit lost when reading the following chapters of the manuscript.

It should be made very clear which role the possible application of the CRISPR/Cas9-technology has in this context. At least this is what I would expect from reading the abstract as it proposes CRISPR/Cas9 to be an important technology contributing to solve the challenges for rice production. Also, the introduction points towards this aspect.

---We are grateful for reviewer’s critical comments. We revised it in Lines 21~23 of the Abstract as follows: This review highlights the mechanisms and optimization of the CRISPR system, and its application to rice crop, including resistance to biotic and abiotic stresses, and improved rice quality and yield.

Personally, I would like to read more about the concrete challenges, and how the current and future trends in CRISPR/Cas9 research in rice can contribute.

I recommend considering concise and easily comprehensible argumentation lines concerning the concrete challenges for rice production and potential solutions. Following a clear argumentation line throughout the manuscript would help the reader to understand the challenges (e.g. in the introduction), leading him/her to why research on certain traits is important and finally to the potential solutions as presented in the manuscript.

---Thank you very much for your critical comments. We have revised the concern in Lines 62~68 of the Introduction as follows: To develop new varieties by CRISPR/Cas9 technology breeders should firstly identify genes that act as negative regulators in yield increase, quality improvement, abiotic stress tolerance, and biotic stress resistance as shown in Table 1. These genes can be applied to produce specific mutations for developing new varieties. In the near future, breeders should use the technique of homology directed repair (HDR) to facilitate their breeding strategies for specific gene of interest. In this review, the current application and future prospects are discussed.

For instance, I had difficulties following the rationale in chapter 3.1., but I consider chapter 3.1. very important for the manuscript. Unfortunately, it is not very useful for the scope of the manuscript in its current form. In my opinion, it should be expanded and specifically explain the challenges of rice transformation and the application of genome editing in rice in more detail.

---We are grateful for reviewer’s critical comments. The first section of “3.1 Rice improvement via CRISPR/Cas9 system” described the general statements on gene editing by CRISPR/Cas9 system. We have described the challenges and application on gene editing to traits of interest from the Line 156.

I very much like the idea of Figure 3, as it nicely explains the steps required to perform genome editing. However, it may be improved significantly; some remarks: Please check the captions in the pictures for spelling and accuracy of information; please rephrase the legend in order to fit the pictures precisely. Please reconsider the following points: caption of picture A, as “gene targeting” might not be the right wording; it is not clear where the rice callus for transformation comes from as this step is missing in the explanation and please rephrase the corresponding legend; picture F: please make the message clear to the reader; picture “I” might be misleading: was it your intention to describe a “super plant” that includes all the mentioned traits?

---I am grateful for your kind comments. We revised it in Figure 3 on page 6 as follows: A, C, F, and I diagrams were improved in Figure 3 according to reviewer’s comments. The Figure legend is described as follows: Figure 3. General strategy for rice development using the CRISPR/Cas9 system. (A) Identify the target gene sequence in rice genome. (B) Design sgRNA and prepare vector construct. (C) After confirming the correct sequence of the construct, it is transferred either by Agrobacterium, biolistic bombardment (gene gun), or protoplast method. The construct is then transformed to rice callus. (D) T0 plants are regenerated from infected callus. (E) Regenerated plants are confirmed through PCR, restriction digestion, and sequencing. (F) In T1 generation, according to Mendelian segregation rules, 25% of plants will be-free edited (null) plants. (G) Rice plants are then screened for desired characteristics. (H) Selected plants are transplanted for field trials. (I) New Rice variety with high yield, high quality trait, disease resistance, and abiotic stress tolerance is then developed by genome editing scientist.

The following chapters contain interesting information and correspond to the scope of the manuscript. I would suggest designing an argumentation line and to balance the amount of the information better. As a reader, I would like to be convinced that the presented traits are indeed selected based on the importance in rice breeding, and it would be interesting to see challenges and success stories in a systematic and easily understandable way. For example, male sterility is important to facilitate rice breeding, which is the tool to solve the challenges in rice production by targeting specific traits. This should be presented in a clear way. Currently, I do not see a rationale behind the selection of papers.

--- Thank you for your critical comments. Reviewer mentioned that “For example, male sterility is important to facilitate rice breeding, which is the tool to solve the challenges in rice production by targeting specific traits. This should be presented in a clear way.” Yes, it may be a good way to give more details on each traits of interest. However, we have provided major information that breeders need to look at the important functional changes by CRISPR/Cas9 mutation and use the information for their breeding programs. There are over 70 genes in Table 1, 2, and 3 that have tested gene functions by knocking out with CRISPR/Cas9 system. I think the purposes of this review article is to provide the functional information of many important genes that regulate traits of interest for breeding program and then breeders can use the information for their specific breeding strategy. We tried to emphasize some genes related to important agronomic traits. If we describe too much details on all genes, the volume of this review article will be too big…

The authors frequently switch between general and specific topics, which makes the text hard to follow for the reader. The text contains redundancies, e.g. lines 46-47. I would also like to mention that Chapter 2 on the general mechanisms of the CRISPR/Cas9 system could be significantly shortened for the benefit of discussing the challenges in rice production in a more detailed way. It would be of value to also focus more on the specificities of using the system in plants. I furthermore recommend taking care of the relevance and accuracy of descriptions (e.g. lines 74 fI agree that it is important to point out the differences between NHEJ and HDR, as this is of major relevance when designing experiments targeting the improvement of plant characteristics. The figure the authors presented is of a general nature and has already been presented in various forms and details in a number of papers. For this reason, it might be redundant to state an original source; however, if the figure has been designed/modified based on a specified source it should be mentioned.

---I am grateful for your critical comments. We modified a sentence in Line 48. We deleted Lines 84~100 from the 1^st submitted paper to short the content. The explanation and comparison of non-homologous end-joining (NHEJ) or homology directed repair (HDR) modifications is described in Lines 115~127 on page 4. We included “(Modified from Doudna JA, Charpentier E, 2014)” in Figure 1 legend at Line 135 on page 4.

Chapter 4: This chapter should contain a discussion on the trends (e.g., can CRISPR/Cas9 target the right traits and thus indeed improve rice productivity?), and the challenges of CRISPR/Cas9 applications in rice breeding. I suggest including a brief analysis of the trends displayed in the previous chapter and how useful they are to enhance rice breeding. Are there traits that have not been targeted so far and what may be the reasons. It would be interesting to read a discussion why CRISPR/Cas9 can make a major contribution and be a significant addition to traditional rice breeding. Like this, you would put Figure 4 into better context. The idea of Figure 4 is appreciated. However, please check it for accuracy, including the legend!

---Thank you for your critical comments. We have modified a large part in Line 551~645. We revised the legend of Figure 4. We also include a paragraph in Line 639~645 as follow: As implications for breeders, to develop new varieties with CRISPR/Cas9 technology by inducing InDel of genes expressing important agronomical traits, breeders first identify genes that act as negative regulators in yield increase, quality improvement, abiotic stress tolerance and biotic stress resistance. They can apply these genes to create specific mutations to develop new varieties. In the near future, breeders should apply aggressively homology-directed repair (HDR) techniques to facilitate breeding strategies for specific genes of interest for developing new varieties.

Remarks on specific content:

Please also be very careful when stating “genome-edited crops do not carry transgenesis DNA…”. This sentence should be rephrased. Although frequently the case, it is not correct to state that genome edited crops are always free of foreign DNA.

---Thank you for your critical comments. We revised it in Line 627 as follows: Many countries formally declare that crops will not be regulated under biosafety legislation if the product of the genome-edited crops do not contain foreign DNA [157].

Please consider that sentences like the ones in 249-252 do not seem to be in the right place.

---Thank you for your kind comments. We deleted the sentences in line 265-269 and wrote a sentence in Lines 264~265 as follows: The CRISPR/Cas9 system technological protocol and its variations are the most dependable and efficient developed for rice production [63].

I would like to see more attention in delivering the messages, to avoid readers spending time to figure out the exact meaning of messages: e.g. line 84: “The CRISPR sequence…” or line 131: “The most direct use…”, etc.

---I am grateful for your critical comments. We deleted the part in Lines 84~100 from the original manuscript that have been mentioned in many other papers.

The following sentences are not accurate or misleading in their current wording: “traditional GMO regulations are circumvented” (lines 138-139), “Genome edited crops do not…” (lines 557-558) and “Transgenic organisms are strictly regulated…” (lines 559-562). These sentences should be reconsidered to provide correct information to the reader. Please also avoid sentences like: “CRISPR/Cas9 system has potential…” (line 142), as they do not deliver relevant information, or rephrase them. It is very relevant though to explain the advantages and the potential of the technology (as in lines 145 ff.).

---Thank you for your critical comments. We revised as follow:

1. Lines 138-139 was changed as follow: “All those activations removed the limitations of standard GMO regulations” in Lines 148-149 on the pages 5.
2. Lines 142.was changed as follow: “CRISPR/Cas9 method is simple, efficient, and precise, it is anticipated to have a significant” in Lines 152~153.
3. Lines 557-558 was changed as follow: “Many countries formally declare that crops will not be regulated under biosafety legislation if the product of the genome-edited crops do not contain foreign DNA” in Lines 627-628.
4. Lines 559-562 was modified as follow: “CRISPR/Cas9 enables the generation of non-GMO mutant plants, an approach that has been widely applied in diverse genomic architectures to study its function as well as its resistance to biotic and abiotic challenges, suitable agronomic and other essential agronomic characteristics, matching with current biosafety regulations for GMO plants” in Lines 632~635 on pages 7.
5. We also include a paragraph in Line 639~645 as follow: As implications for breeders, to develop new varieties with CRISPR/Cas9 technology by inducing InDel of genes expressing important agronomical traits, breeders first identify genes that act as negative regulators in yield increase, quality improvement, abiotic stress tolerance and biotic stress resistance. They can apply these genes to create specific mutations to develop new varieties. In the near future, breeders should apply aggressively homology-directed repair (HDR) techniques to facilitate breeding strategies for specific genes of interest for developing new varieties.

Remarks on spelling and style:

Concerning spelling and English style, there are many missing definite/indefinite articles. In addition, there is a remarkable amount of very long sentences that are hard to comprehend.

The spelling and font type of gene, species, and mutant names (e.g., italic; use of capital and small letters;…) has to be carefully checked throughout the manuscript.

---I am grateful for your critical comments. We revised all throughout the manuscript

Spelling of CRISPR/Cas9 needs to be unified.

---I am grateful for your critical comments. We checked and revised all throughout the manuscript.

Is there something missing in the sentence “These findings…” (lines 298-299)?

---I am grateful for your critical comments. We revised it in Line 320~321 of the manuscript as follows: These findings in T2 generation suggest that CRISPR/Cas9 might be an effective tool for increasing rice salt [75] (Table 2).

Table 1. Check spelling and font types.

---I am grateful for your detailed comments. We checked and revised all in Table 1, 2, and 3.

Reviewer 2 Report

This review focusses on using CRSPR/Cas technology in rice. There are quite a few reviews on genome editing in plants in general and also restricted to specific plants and traits. Another review is only justified if it really adds some new insight and/or gives a clearly overview what has been achieved so far.

In my opinion the present manuscript does not meet these criteria. The main critics are as follows:

1. The technology of CRSPR/Cas has been reviewed numerous times and it is not justified to review it again by using 2 pages.
2. In 7 subchapters in chapter 3.1 (Rice improvements…) a summary is given and it is referred to Table 1 extending over 4 pages. This table is organized in several sectors whose attribution to the subchapters is absolutely unclear, as several sectors, but not all, refer to one subchapter and the order differs from the main text. This unorganized presentation is not at all acceptable for a review.

In conclusion, this chaotic presentation does not help the reader to get a concise overview. It is not the reviewer`s job to guide the authors to make a well-organized review article.

Author Response

Responses to Reviewer 2:

We are grateful for the reviewer on valuable comments and suggestions for the manuscript. We believe that the comments have identified important areas that required improvement. The revisions or answers are as follows:

This review focusses on using CRSPR/Cas technology in rice. There are quite a few reviews on genome editing in plants in general and also restricted to specific plants and traits. Another review is only justified if it really adds some new insight and/or gives a clearly overview what has been achieved so far.

In my opinion the present manuscript does not meet these criteria. The main critics are as follows:

The technology of CRSPR/Cas has been reviewed numerous times and it is not justified to review it again by using 2 pages.

---I am grateful for your critical comments. We have revised it in Line 62~68 of the manuscript and shortened some parts unnecessary in Chapter 2.  We deleted the part in Lines 84~100 from the original manuscript that have been mentioned in other papers. We have improved large parts in chapter 4 in Lines 551~645.

In 7 subchapters in chapter 3.1 (Rice improvements…) a summary is given and it is referred to Table 1 extending over 4 pages. This table is organized in several sectors whose attribution to the subchapters is absolutely unclear, as several sectors, but not all, refer to one subchapter and the order differs from the main text. This unorganized presentation is not at all acceptable for a review.

In conclusion, this chaotic presentation does not help the reader to get a concise overview. It is not the reviewer`s job to guide the authors to make a well-organized review article.

---Thank you for your critical comments. We have divided Table 1 into three Tables according to the agronomic traits group. They are presented Table 1 on page 10, Table 2 on page 13, and Table 3 on page 16.

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper "Research Trends and Challenges of Using CRISPR/Cas9 for Improving Rice Productivity" is very interesting and important.

All Tables and Figures are perfect.

In Review papers very important is meta-analysis.
In this paper lack of meta-analysis.

Paper needs minor revision.

Author Response

Responses to Reviewer 3:

We are grateful for the reviewer on valuable comments and suggestions for the manuscript. We believe that the comments have identified important areas that required improvement. The revisions or answers are as follows:

The paper "Research Trends and Challenges of Using CRISPR/Cas9 for Improving Rice Productivity" is very interesting and important.

All Tables and Figures are perfect.

In Review papers very important is meta-analysis.
In this paper lack of meta-analysis.

Paper needs minor revision.

---I am grateful for your critical comments. We have revised it in Line 62~68 of the manuscript and shortened some parts unnecessary in Chapter 2.  We deleted the part in Lines 84~100 from the original manuscript that have been mentioned in other papers. We have improved large parts in chapter 4 in Lines 551~645.

We have improved Figure 3 to provide the detailed information on general strategy for rice development using the CRISPR/Cas9 system.

We have divided Table 1 as three Tables according to the agronomic traits group. They are presented Table 1 on page 10, Table 2 on page 13, and Table 3 on page 16.

Author Response File: Author Response.pdf

Reviewer 4 Report

1. In the present review article, the authors summarize the status of genome editing approaches that have been utilized for rice improvement, as well as the potential future challenges for the widespread adoption of this technology.  More specifically, they provide a brief but concise summary of the science behind the CRISPR/Cas9 system and subsequently list a series of studies that CRIPSR/Cas9 has been used for grain yield increase, improvements in quality and nutrition, as well as enhancement of plants against abiotic and biotic stresses, and other areas that could facilitate improvements in hybrid breeding through the generation of male stripe lines.  The section with challenges and future prospects is also specific.  Overall, I think it is a comprehensive review article that presents a broad overview of the activities for the utilization of CRISPR/Cas9 for rice improvement.  The only thing that I believe is missing is a section of conclusions/discussion that the authors could provide some generalized observations from the studies that they included in their review (for example, the pattern for the selection of promoters that drive the expression of genome editing components as presented in Table 1. Is pretty clear, similar to the transformation methods and the type of mutations).
   

Author Response

Responses to Reviewer 4:

We are grateful for the reviewer on valuable comments and suggestions for the manuscript. We believe that the comments have identified important areas that required improvement. The revisions or answers are as follows:

2. In the present review article, the authors summarize the status of genome editing approaches that have been utilized for rice improvement, as well as the potential future challenges for the widespread adoption of this technology.  More specifically, they provide a brief but concise summary of the science behind the CRISPR/Cas9 system and subsequently list a series of studies that CRIPSR/Cas9 has been used for grain yield increase, improvements in quality and nutrition, as well as enhancement of plants against abiotic and biotic stresses, and other areas that could facilitate improvements in hybrid breeding through the generation of male stripe lines.  The section with challenges and future prospects is also specific.  Overall, I think it is a comprehensive review article that presents a broad overview of the activities for the utilization of CRISPR/Cas9 for rice improvement. 
3. The only thing that I believe is missing is a section of conclusions/discussion that the authors could provide some generalized observations from the studies that they included in their review (for example, the pattern for the selection of promoters that drive the expression of genome editing components as presented in Table 1. Is pretty clear, similar to the transformation methods and the type of mutations).

---We are grateful for your critical comments. We have revised it in Line 62~68 of the manuscript and shortened some parts unnecessary in Chapter 2.  We deleted the part in Lines 84~100 from the original manuscript that have been mentioned in other papers. We have improved large parts in chapter 4 and chapter 5 in Lines 551~659.

We have improved Figure 3 to provide the detailed information on general strategy for rice development using the CRISPR/Cas9 system.

We have divided Table 1 as three Tables according to the agronomic traits group. They are presented Table 1 on page 10, Table 2 on page 13, and Table 3 on page 16.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revisions, in my opinion, have significantly improved the manuscript. I believe that it is much easier to read and understand, and that it confers a clearer message than the previous version.

However, a few concerns remain, which I would like to ask the authors to consider carefully.

Please reconsider and improve the explanation lines 101-117: I suggest to reconsider which information is essential and please be accurate when describing this information. Also, I suggest to reconsider the order of sentences to improve comprehensibility.

Lines 560-575: Also the knowledge on gene function and whether species can be readily transformed are limitations. You may add this.

Line 557: The term “top types” has not been introduced. Please reconsider/use another wording

Line 527: alternative: “more precision is necessary”

Lines 637-643: You may also add that limiting factors are the functional annotation of genes, their accessibility to be altered by the available technologies, and that the application of HDR technologies in plants is still technically limited. It is sufficient to use HDR here, as the abbreviation has been introduced earlier.

Please check the following sentences that seem to be incomplete:

Lines 34-35

Lines 152ff

Lines 264-265: misleading sentence

Lines 363-364: please check; could be removed?

Lines 557-559

Rephrasing:

Lines: 327-331: Sentence too long. Please rephrase!

Lines 406-409: Please check this sentence and rephrase it to fit it into context.

Figure 3, Legend:

D: “Infected” does not seem to be appropriate.

F: “will be-free edited”? Please revise.

I: Check spelling: upper/lower case letters)

Figure 4, Legend:

D: Spelling: through; “chemical radiation”: Either radiation or chemical substances my induce mutations. Please reconsider and rephrase, e.g. “radiation and chemical mutagenesis”.

Minor remarks:

Line 76: Please remove: “then”

Lines 247-248: Move to the next paragraph?

Line 336: Replace: “were” – “was”

Line 372: I suggest to use “heat stress tolerance”

Line 641: I suggest to remove the term “aggressively”

Line 651: I suggest to remove “ORF”

Spelling:

Throughout the manuscript: Please check genes, mutant lines, Agrobacterium… for the correct use of the italic font; check the spelling of “indels”.

Page 3: Please check again the spelling of CRISPR/Cas9

Line 110: Pyogenes (lower case)

Line 555: genome

Figure 1: Non-homologous end joining

Figure 3: Captions: use of upper or lower case? Please unify.

Author Response

Reviewer 1

We are grateful for the reviewer’s valuable comments and suggestions for the manuscript. We believe that the comments have identified important areas that required improvement. The revisions or answers are as follows:

The revisions, in my opinion, have significantly improved the manuscript. I believe that it is much easier to read and understand, and that it confers a clearer message than the previous version.

However, a few concerns remain, which I would like to ask the authors to consider carefully.

Please reconsider and improve the explanation lines 101-117: I suggest to reconsider which information is essential and please be accurate when describing this information. Also, I suggest to reconsider the order of sentences to improve comprehensibility.

---Thank you for your kind comments. We have modified the part on Lines 80~87 on page 3.

Lines 560-575: Also the knowledge on gene function and whether species can be readily transformed are limitations. You may add this.

---I appreciate the reviewer’s comments. We revised it in Line 572~576 as follows: First of all, breeders should set up the efficient transformation system for CRISPR/Cas9-Target gene vector to develop enough number of transgenic plants and to confirm the gene edited plants. This application should be set up well for each target plant species. The success of creating the gene edited plants may depend on firstly in this step.

Line 557: The term “top types” has not been introduced. Please reconsider/use another wording

--- Thank you for your kind comments. We have modified it in Line 531~533 as follows: CRISPR/Cas9 Genome editing can improve a characteristic by accurately altering and rapidly rearranging chromosomes in one of the best breeding strategies.

Line 527: alternative: “more precision is necessary”

--- Thank you for your kind comments. We have revised it in Line 500~502 as follows: HDR is a technology that requires donor DNA with high homology to replace key SNPs or specific regions in genomic DNA, and has not yet been generalized in higher plants.

Lines 637-643: You may also add that limiting factors are the functional annotation of genes, their accessibility to be altered by the available technologies, and that the application of HDR technologies in plants is still technically limited. It is sufficient to use HDR here, as the abbreviation has been introduced earlier.

--- Thank you for your kind comments. We have revised it in Line 607~613 as follows: As implications for breeders, to develop new varieties with genes expressing important agronomical traits by CRISPR/Cas9 technology, breeders first identify genes that act as factors in yield increase, quality improvement, abiotic stress tolerance and biotic stress resistance. They can apply these genes to create specific mutations to develop new varieties. In the near future, breeders should apply homology-directed repair (HDR) techniques to facilitate breeding strategies for specific genes of interest for breeding new varieties.

Please check the following sentences that seem to be incomplete:

Lines 34-35: ---We have rewritten it in Line 32~33 as follows: Climate change is also projected to increase global temperatures by 2 degrees Celsius over the next 30 years.

Lines 152: ---We have rewritten it in Line 130~132 as follows: As the CRISPR/Cas9 method is simple, efficient, and precise, it is anticipated to have a significant effect on plant biology and crop breeding.

Lines 264-265: misleading sentence: ---We have rewritten it in Line 242~243 as follows: The advantages of CRISPR/Cas9 technology and its efficiency can be applied as the most dependable and efficient way for new rice breeding [67].

Lines 363-364: please check; could be removed?

--- Thank you for your kind comments. We revised it in Line 379 as follows: As a result, OsAnn3 was thought to have a function in rice cold tolerance [109] (Table 1).

Lines 557-559

Rephrasing: We revised it in Line 534~536 as follows: The comparisons of traditional breeding and the modern breeding methods for rice improvement are described in detail as shown in Figure 4.

@ We have moved two parts from “3.1.6 and 3.1.7” to “3.1.3 and 3.1.4”, which are positioned in Line 273~313 on page 9. And also Table 2 & 3 are combined as Table 2 for abiotic and biotic stress tolerance.

Lines: 327-331: Sentence too long. Please rephrase!

We revised it in Line 341-347 as follows: Rice osera1 mutant lines with CRISPR/Cas9-induced frameshift mutations in OsERA1 revealed comparable leaf growth to wild-type (WT) plants. These mutant lines show improved main root development than the WT, through stomatal regulation, these osera1 mutant lines also had better sensitivity to ABA and drought stress.

Lines 406-409: Please check this sentence and rephrase it to fit it into context.

We revised it in Line 422-426 as follows: Recent results on pathogen-associated to biomolecular analysis in rice blast disease are discussed. Pyramiding several R genes and other methods such as host-induced gene silencing, cross-species transformation, and gene editing might be useful for creating broad-spectrum resistant varieties Recent results on pathogen-associated to biomolecular analysis in rice blast disease are discussed. Pyramiding several R genes and other methods such as host-induced gene silencing, cross-species transformation, and gene editing might be useful for creating broad-spectrum resistant varieties.

Figure 3, Legend: Thank you very much for your kind comments…

D: “Infected” does not seem to be appropriate. --- Modified to ‘transformed’

F: “will be-free edited”? Please revise.--- Modified to ‘will be gene-edited null plants’

I: Check spelling: upper/lower case letters) --- Modified to ‘rice’

Figure 4, Legend:

D: Spelling: through; “chemical radiation”: Either radiation or chemical substances may induce mutations. Please reconsider and rephrase, e.g. “radiation and chemical mutagenesis”.

--- Modified to ‘radiation and chemical mutagenesis’

Minor remarks:

Line 76: Please remove: “then”  --- Thanks, we deleted it.

Lines 247-248: Move to the next paragraph?  ---Thanks, we have moved it.

Line 336: Replace: “were” – “was” ---Thanks, we have corrected it.

Line 372: I suggest to use “heat stress tolerance”  ---Thanks, we have corrected it.

Line 641: I suggest to remove the term “aggressively” --- Thanks, we deleted it.

Line 651: I suggest to remove “ORF”  --- Thanks, we deleted it.

Spelling:

Throughout the manuscript: Please check genes, mutant lines, Agrobacterium… for the correct use of the italic font; check the spelling of “indels”.  ---Thanks, we have corrected it.

Page 3: Please check again the spelling of CRISPR/Cas9  ---Thanks, we have corrected it.

Line 110: Pyogenes (lower case) ---Thanks, we have corrected it.

Line 555: genome ---Thanks, we have corrected it.

Figure 1: Non-homologous end joining ---Thanks, we have corrected it.

Figure 3: Captions: use of upper or lower case? Please unify. ---Thanks, we have corrected it.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have separated the original Table into 3 Tables. However withinin each table the order of functional parameters differs from the order discussed in the text.

In my opinion the manuscript is still most chaotic and thus does not meet the basic standards for a review article.

Author Response

Reviewer 2

We are grateful for the reviewer’s valuable comments and suggestions for the manuscript. We believe that the comments have identified important areas that required improvement. The revisions or answers are as follows:

The authors have separated the original Table into 3 Tables. However within in each table the order of functional parameters differs from the order discussed in the text.

In my opinion the manuscript is still most chaotic and thus does not meet the basic standards for a review article.

--- I am grateful for your critical comments. We have revised and re-arranged the manuscript follow your advice as below:

1. We re-arranged two parts from “3.1.6 and 3.1.7” to “3.1.3 and 3.1.4”, which are positioned in Line 273~313 on page 9.
2. Table1 & Table2, were arranged in order discussed with the text in the manuscript
3. Table 2 & 3 are combined as Table 2 for abiotic and biotic stress tolerance.
4. We have also changed the reference numbers from the No. 53 (Lines 915) to No. 156 (line 1191)

Author Response File: Author Response.pdf