CRISPR/Cas in Grapevine Genome Editing: The Best Is Yet to Come

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors provide a comprehensive overview of the advances in grapevine genome editing achieved over the last decade. Moreover, they describe the development and advances in CRISPR/Cas9 and its orthologs and variants. The applications of genome editing in grapevine are then summarized. The final sections focus on the discussion of the challenges as well as the possible strategies that could prompt the improvement of editing in grapevine.

 

The review is of interest and very pertinent to the special issue proposed; however, I advise that the manuscript should be revised prior publication.

 

My recommendations:

-              The delivery of CRISPR/Cas reagents into the plant cells and their regeneration are the MAIN CHALLENGES of the application of genome editing in plants in general and even more in recalcitrant species like the grapevine. This is especially crucial when the goal is to generate a DNA-free edited grapevine and to avoid chimerism (that certain delivery systems cannot guarantee). The author should greatly expand this section in their manuscript, presenting the many different delivery methods, their pros and cons, and how those apply to plants and, ultimately, to the grapevine. Some of these concepts are discussed in “5.4. Generating transgene-free edited grapevine plants”, but these topics should be more organically organized in the text to emphasize their significance. 

-              The “future perspectives” part should also deal with how genome edited plants are regulated worldwide. The EU has been discussing extensively the regulation of these improved plants (categorized as NGT1 plants) and is yet to provide a final guideline. The position that the EU will take on this regard is of particular importance for the grapevine since the relevance of the wine sector in the Old-World economy. The final result will likely have a driving effect on other indecisive countries. All these considerations and many more could be included in an additional subsection of section 5. 

-              Minor edit: remember to remove lines 29-34.

 

 

Author Response

The authors provide a comprehensive overview of the advances in grapevine genome editing achieved over the last decade. Moreover, they describe the development and advances in CRISPR/Cas9 and its orthologs and variants. The applications of genome editing in grapevine are then summarized. The final sections focus on the discussion of the challenges as well as the possible strategies that could prompt the improvement of editing in grapevine.The review is of interest and very pertinent to the special issue proposed; however, I advise that the manuscript should be revised prior publication.

 

My recommendations:

Comments 1: The delivery of CRISPR/Cas reagents into the plant cells and their regeneration are the MAIN CHALLENGES of the application of genome editing in plants in general and even more in recalcitrant species like the grapevine. This is especially crucial when the goal is to generate a DNA-free edited grapevine and to avoid chimerism (that certain delivery systems cannot guarantee). The author should greatly expand this section in their manuscript, presenting the many different delivery methods, their pros and cons, and how those apply to plants and, ultimately, to the grapevine. Some of these concepts are discussed in “5.4. Generating transgene-free edited grapevine plants”, but these topics should be more organically organized in the text to emphasize their significance. 

Response 1: Thanks for the suggestion. We expanded the “delivery of CRISPR/Cas reagents” section  and discussed different delivery methods in the manuscript as suggested (page 14).

Comments 2: The “future perspectives” part should also deal with how genome edited plants are regulated worldwide. The EU has been discussing extensively the regulation of these improved plants (categorized as NGT1 plants) and is yet to provide a final guideline. The position that the EU will take on this regard is of particular importance for the grapevine since the relevance of the wine sector in the Old-World economy. The final result will likely have a driving effect on other indecisive countries. All these considerations and many more could be included in an additional subsection of section 5. 

Response 2: Thanks for the suggestion. The content about regulation of gene-edited plants had been added in the manuscript as an additional section 5 as suggested by the reviewer (page 15).

Comments 3: Minor edit: remember to remove lines 29-34.

Response 3: The sentences had been removed from the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The article includes a detailed description of the different tools that can now be used to apply gene editing in plants, highlighting the different opportunities offered by the different GE tools and also the complexity in identifying the most appropriate tool depending on the type of modification expected. This complexity should be better highlighted in the discussion on the application of this technology.

It reports that mainly fairly well-known genes tested in other plants are now tested also in grape, and almost exclusively on Thompson seedless, the variety known as the easiest to transform with agrobacterium. Therefore how far this technology can be really expanded to all other most important grape cutiviars?

Most of the experiment on protoplast report results on gene editing on callus lines and not plants, such as what reported in citation 17. The bottle neck of plant regeneration from callus derived from edited protoplasts should be evidenced.

It is clear that EC regeneration and grape transformation are strictly genotype dependent, and specific protocols must be developed for each cultivar. Regeneration from calluses from protoplasts is even more difficult. These aspects should be better highlighted in the article, as well as mentioning the technologies that now seem to allow us to solve this problem. In fact, about morphogenic regulators, they can contribute to the increase of the regeneration efficiency but to be understand how far they can affect the phenotype of the regenerated plants.

Most of the results now available are derived by agro transformation, so fully considered as GMO, the proposed alternative solutions can be acceptable but how far easily applicable in all grape varieties.  

The work describes the different gene editing techniques now known making references to those already tested in grape, but more clear indication should be given on which techniques may be more efficient for the main objectives that can be foreseen for the genetic improvement of the grape.

The article totally lacks considerations on the current regulatory problems of the different GE products (for example differentiating products from transformation with agro and those from protoplasts) and the acceptability by the market and the consumer.

Important references on regeneration/transformation issues, such as biosafety and rules are missing.

Comments on the Quality of English Language

A revision is needed.

Author Response

Comments 1: The article includes a detailed description of the different tools that can now be used to apply gene editing in plants, highlighting the different opportunities offered by the different GE tools and also the complexity in identifying the most appropriate tool depending on the type of modification expected. This complexity should be better highlighted in the discussion on the application of this technology.

Response 1: Thanks for the suggestion. As pointed by the reviewer, we described different tools here that we hope they can be used in grapevine in the future. Of course, some of them have already been applied in grapevine, and most of them should be tested and experimentally confirmed. We agree with the reviewer that the complexity would be better highlighted in the  discussion on the application of CRISPR technology. In fact, applications of CRISPR in plants have been reviewed many times these years, and we believe readers can easily get the information from these reviews. Here we just try to focus on grapevine genome editing, so we did not discuss more about the applications of this technology.

 

Comments 2: It reports that mainly fairly well-known genes tested in other plants are now tested also in grape, and almost exclusively on Thompson seedless, the variety known as the easiest to transform with agrobacterium. Therefore how far this technology can be really expanded to all other most important grape cutiviars?

Response 2: As we have discussed in challenges and future perspectives (page 15 and 17), the bottleneck in grape genome editing, or rather, grapevine genetic transformation, is species- or genotype-dependent. The problem is that it is not easy to get available explants, generally the embryogenic callus, from most important grape cultivars. Thus, the transformation of grapevine is the MAIN CHALLENGE of the applications of CRISPR technology in grapevine. We do not think it would be a problem if stable transformation system has been established for other important grape cultivars.

 

Comments 3: Most of the experiment on protoplast report results on gene editing on callus lines and not plants, such as what reported in citation 17. The bottle neck of plant regeneration from callus derived from edited protoplasts should be evidenced.

Response 3: According to the reports (Najafi et al., 2022; Scintilla et al., 2022; Tricoli et al., 2023), it is possible to get edited plants from edited protoplasts, though the difficulty in regeneration from protoplasts is generally not emphasized in these studies. We addressed this issue in our revised manuscript (page 14).

 

Comments 4: It is clear that EC regeneration and grape transformation are strictly genotype dependent, and specific protocols must be developed for each cultivar. Regeneration from calluses from protoplasts is even more difficult. These aspects should be better highlighted in the article, as well as mentioning the technologies that now seem to allow us to solve this problem. In fact, about morphogenic regulators, they can contribute to the increase of the regeneration efficiency but to be understand how far they can affect the phenotype of the regenerated plants.

Response 4: Thanks for the suggestion. We had revised our manuscript accordingly (page 14). We also noted the possible adverse effect when using morphogenic regulators, and corresponding solutions were also mentioned in the manuscript (page 15).

 

Comments 5: Most of the results now available are derived by agro transformation, so fully considered as GMO, the proposed alternative solutions can be acceptable but how far easily applicable in all grape varieties.  

Response 5: The core of this question is same to that presented in comment 2. Gene-edited plants derived from edited protoplasts is a promising way to solve the GMO concerns in the future. However, it is not possible to achieve this goal currently due to the lack of ECs in most of grape varieties. The problem is also mentioned in the manuscript (page 15 and 17).

 

Comments 6: The work describes the different gene editing techniques now known making references to those already tested in grape, but more clear indication should be given on which techniques may be more efficient for the main objectives that can be foreseen for the genetic improvement of the grape.

Response 6:  Thanks for the suggestion. We had added the conclusive descriptions in the manuscript in page 7.

 

Comments 7: The article totally lacks considerations on the current regulatory problems of the different GE products (for example differentiating products from transformation with agro and those from protoplasts) and the acceptability by the market and the consumer.

Response 7:  Thanks for the suggestion. An additional section 5 about regulation of gene-edited plants had been added in the manuscript (page 15).

 

Comments 8: Important references on regeneration/transformation issues, such as biosafety and rules are missing.

Response 8: The section about transformation/regeneration had been expanded (page 14).

Reviewer 3 Report

Comments and Suggestions for Authors

In this review, the authors provide a comprehensive analysis of the advances in grapevine genome editing via CRISPR technology to accelerate trait improvement. Besides, the authors review the challenges in grapevine genome editing and the possible strategies to face them. 

I have some remarks:

Abstract. In this sentence: “Advances in grapevine genome editing have been achieved these years, and the use of CRISPR technology promises to accelerate trait improvement” Are there other techniques to edit grapevine besides CRISPR? Improve the sentence to indicate “genome editing” or only “trait improvement”

Line 29-34 Remove the paragraph 

Line 43-47. I consider that if the ZFNs and TALENs strategies have not been used in grapevine genetic editing it could be better not mention them in the introduction. If so, please indicate and discuss them. An option could be review the traditional breeding method to trait improvement and discuss the advantages of CRISPR technology. 

Line 83-84. The classification and introduction given in subtitle 2 does not match with the next subtitle (2.1). You mention first the CRISPR systems: class 1 and 2 systems and the types of target sequences: DNA-targeting and RNA-targeting. However the next subtitle starts with class 2 type II. What about starting with class 1? Please order the ideas according to the first classification that you provide. 

Additionally an image explaining the classification of CRISPR technology will be useful. 

Line 117-118. Introduce the meaning of “N” in PAM variants

Line 136. Does all the information provided in this section refer to grapevine? Indicate the plants used in those experiments and if then could be used in grapevine 

Line 174. At this point you have already mentioned several times the abbreviation of the nucleotides without explaining their meaning. I refer to this sentence: “deaminate cytosine into uracil (U)”

Line 300. Until line 300 I noticed that the information is not related to grapevine. There are several articles explaining CRISPR bases and general. What is new in this review. Discuss how this information is related to grapevine editing. 

Line 308-324. This section is really important for the goal of the review. Can you describe and analyze it? 

Table 1. Please, add a column indicating the advantage/results of modifying the corresponding trait that you mention. 

 

Conclusions. Add the applications of CRISPR genome made in grapevin

e and the traits modified

Comments on the Quality of English Language

Minor editing required 

Author Response

Reviewer 3:

In this review, the authors provide a comprehensive analysis of the advances in grapevine genome editing via CRISPR technology to accelerate trait improvement. Besides, the authors review the challenges in grapevine genome editing and the possible strategies to face them. I have some remarks:

Comments 1: Abstract. In this sentence: “Advances in grapevine genome editing have been achieved these years, and the use of CRISPR technology promises to accelerate trait improvement” Are there other techniques to edit grapevine besides CRISPR? Improve the sentence to indicate “genome editing” or only “trait improvement”

Response 1: We had corrected this description as “Advances in grapevine genome editing have been achieved by using CRISPR technology these years, which promises to accelerate trait improvement in grapevine.”

Comments 2: Line 29-34 Remove the paragraph.

Response 2: The paragraph had been removed.

Comments 3: Line 43-47. I consider that if the ZFNs and TALENs strategies have not been used in grapevine genetic editing it could be better not mention them in the introduction. If so, please indicate and discuss them. An option could be review the traditional breeding method to trait improvement and discuss the advantages of CRISPR technology. 

Response 3: ZFNs and TALENs are two genome editing tools before the emergence of CRISPR. Here we just briefly introduce these two tools to show the advantage of CRISPR technology. We also mentioned the disadvantages of conventional breeding in the manuscript (page 2).

Comments 4:  Line 83-84. The classification and introduction given in subtitle 2 does not match with the next subtitle (2.1). You mention first the CRISPR systems: class 1 and 2 systems and the types of target sequences: DNA-targeting and RNA-targeting. However the next subtitle starts with class 2 type II. What about starting with class 1? Please order the ideas according to the first classification that you provide. 

Response 4: Sorry for the confusing descriptions. We had corrected the descriptions in the manuscript. CRISPR systems can be classified into two classes (class 1 and 2) based on the effector proteins. In addition, according to the types of target sequences, CRISPR systems can be generally divided into DNA-targeting and RNA-targeting systems (page2). Here we discussed the systems according to the latter classification.

Comments 5: Additionally an image explaining the classification of CRISPR technology will be useful. 

Response 5: Addition image (figure 1) had been added as suggested (page 2).

Comments 6: Line 117-118. Introduce the meaning of “N” in PAM variants.

Response 6:The meaning of N had been indicated in the manuscript (page 3).

Comments 7: Line 136. Does all the information provided in this section refer to grapevine? Indicate the plants used in those experiments and if then could be used in grapevine 

Response 7: This section is a general description of different CRISPR tools from mammalian cells, plants (Arabidopsis, rice, etc.), and bacteria. In theory, all these CRISPR systems could be applied and tested in grapevine. The confirmed ones had been discussed later in the manuscript in grapevine genome editing.

Comments 8:Line 174. At this point you have already mentioned several times the abbreviation of the nucleotides without explaining their meaning. I refer to this sentence: “deaminate cytosine into uracil (U)”

Response 8: In general, it is easy to understand the meaning of nucleotides referred to by the reviewer. The uncommon nucleotides are indicated in the manuscript.

Comments 9: Line 300. Until line 300 I noticed that the information is not related to grapevine. There are several articles explaining CRISPR bases and general. What is new in this review. Discuss how this information is related to grapevine editing. 

Response 9: The information is a description of different tools that can now be used to apply gene editing in plants, highlighting the different opportunities offered by the different GE tools. In theory, all these CRISPR systems could be tested and applied in grapevine. In addition, some of these tools that have already been applied in grapevine are mentioned in this section.  

Comments 10: Line 308-324. This section is really important for the goal of the review. Can you describe and analyze it? 

Response 10: Thanks for the suggestion. We had revised our manuscript accordingly (page 7-8).

Comments 11: Table 1. Please, add a column indicating the advantage/results of modifying the corresponding trait that you mention. 

 Response 11: Thanks for the suggestion. We had added a column accordingly in the table (page 9).

Comments 12: Conclusions. Add the applications of CRISPR genome made in grapevine and the traits modified

 Response 12: Thanks for the suggestion. We had revised our manuscript accordingly (page 18).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed my concerns and implemented the manuscript. I consider this new version of the manuscript suitable for publication in Horticulturae.