Genetic Engineering in Bacteria, Fungi, and Oomycetes, Taking Advantage of CRISPR

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review examines the application of the CRISPR Cas9 system in various microorganisms: bacteria, fungi, and oomycetes. Both the mechanisms of action at the molecular level and the applications are described in detail. The article is very informative, easy to read, and enriches the reader with a lot of new information.

I have a few suggestions for improvements:

1. The authors should rearrange the whole article and put the figures in their place in the text to make it easier to understand. Please note that moving the tables will change the numbering of the references.

2. Since the whole review is only about CRISPR Cas9, this method should be presented in the title. The current title is very general and does not reflect the content of the article.

3. In Table 2, it is good to indicate the strain in addition to the species of microorganisms, since many of the qualities (especially for bacteria) are strain-specific.

Author Response

We sincerely thank Reviewer 1 for his/her positive comments and constructive criticism that have greatly improved our manuscript. For detailed responses to each point raised, please see below.

 

Comments 1: [The authors should rearrange the whole article and put the figures in their place in the text to make it easier to understand. Please note that moving the tables will change the numbering of the references.]

Response 1: [Agree. We have rearranged the whole article and put the figures in their place in the text to make it easier to understand. More details can be found in our revised manuscript. Thank you!]

 

Comments 2: [Since the whole review is only about CRISPR Cas9, this method should be presented in the title. The current title is very general and does not reflect the content of the article.

Response 2: [Agree. We have revised the title to “Genetic Engineering in Bacteria, Fungi, and Oomycetes taking advantage of CRISPR”. More details can be found in our revised manuscript. Thank you!]

 

Comments 3: [In Table 2, it is good to indicate the strain in addition to the species of microorganisms, since many of the qualities (especially for bacteria) are strain-specific.]

Response 3: [Agree. We have further indicated the strain information in Table 2. More details can be found in our revised manuscript. Thank you!]

 

Reviewer 2 Report

Comments and Suggestions for Authors

The review covers a vast amount of material, but clearer organization and deeper exploration of key challenges would greatly improve its effectiveness. More detailed case studies and a fuller discussion of the ethical and technical hurdles would give the review more depth and practical relevance. The comparison between organisms needs to be sharper and more data-driven. A table or figure summarizing key differences would help readers grasp the distinctions more easily. Some points to be considered are noted below:

While CRISPR/Cas9 is mentioned throughout, there’s no thorough comparison of why certain methods work better in one organism versus another or a detailed discussion of alternative technologies that may be more suitable in cases where CRISPR shows limitations.

The use of Table 1 is referenced but not explained in enough depth, leaving out critical quantitative comparisons of transformation efficiencies or success rates between these organisms. Including more detailed performance data, perhaps in terms of gene editing success or precision metrics, would strengthen the analysis.

The review emphasizes the challenges in fungi and oomycetes but fails to equally highlight specific bacterial engineering challenges, particularly beyond HGT concerns. Given the prominence of CRISPR in bacterial research, more critical evaluation of limitations and future enhancements in bacterial genetic engineering would make the comparison more balanced.

Each organism (bacteria, fungi, and oomycetes) faces unique challenges, but the discussion of these challenges feels rushed or incomplete. Provide a deeper examination of the technical limitations—such as off-target effects in fungi, transformation difficulties in oomycetes, or resistance to CRISPR technologies—and offer insights into ongoing research aimed at overcoming these issues.

There is some mention of ecological impacts and horizontal gene transfer risks, but this discussion could be more prominent throughout. Engage with the broader ethical and ecological debates in biotechnology, including public concern about GMOs, unintended ecological consequences, and long-term risks of deploying genetically modified organisms.

The comparative analysis should draw clearer distinctions between the organisms. Right now, the analysis hints at differences but could be more explicit. Discuss, for example:

• How bacteria’s single-cell simplicity makes them easier to genetically modify.
• How fungi’s multicellular complexity creates barriers to transformation.
• How oomycetes’ evolutionary distinctness presents unique challenges in manipulating their genomes.

A table or figure comparing key genetic engineering metrics (e.g., transformation efficiency, off-target risks, ecological concerns) across bacteria, fungi, and oomycetes could help visually reinforce these differences.

Expand on how specific Cas9 variants or delivery methods (e.g., nanoparticles) will address the existing barriers in each organism.

Explore the future potential of synthetic biology with more detail—what practical applications might emerge from constructing synthetic gene networks or microbial factories?

Elaborate on the integration of multi-omics approaches (e.g., transcriptomics, proteomics) in discovering new gene targets or improving pathogen resistance, particularly in fungi and oomycetes.

 

Highlight emerging tools (like CRISPR/Cas12a or base editors) in greater detail for their specific advantages in addressing the genome-editing challenges in fungi and oomycetes.

Author Response

We sincerely thank Reviewer 2 for his/her positive comments and constructive criticism that have greatly improved our manuscript. For detailed responses to each point raised, please see below.

 

Comments 1: [The review covers a vast amount of material, but clearer organization and deeper exploration of key challenges would greatly improve its effectiveness. More detailed case studies and a fuller discussion of the ethical and technical hurdles would give the review more depth and practical relevance. The comparison between organisms needs to be sharper and more data-driven. A table or figure summarizing key differences would help readers grasp the distinctions more easily]

Response 1: [Agree. We have added more detailed case studies and a fuller discussion of the ethical and technical hurdles to improve our manuscript’s depth and practical relevance. We also revised the manuscript to make the comparison between organisms sharper and more data-driven. A new table [Table 3] summarizing key differences was added accordingly. More details can be found in our revised manuscript. Thank you!]

 

Comments 2: [While CRISPR/Cas9 is mentioned throughout, there’s no thorough comparison of why certain methods work better in one organism versus another or a detailed discussion of alternative technologies that may be more suitable in cases where CRISPR shows limitations.]

Response 2: [Agree. We have further added a thorough comparison accordingly. More details can be found in our revised manuscript. Thank you!]

 

Comments 3: [The use of Table 1 is referenced but not explained in enough depth, leaving out critical quantitative comparisons of transformation efficiencies or success rates between these organisms. Including more detailed performance data, perhaps in terms of gene editing success or precision metrics, would strengthen the analysis.]

Response 3: [Agree. We have further added more critical quantitative comparisons accordingly (Table 3). More details can be found in our revised manuscript. Thank you!]

 

Comments 4: [The review emphasizes the challenges in fungi and oomycetes but fails to equally highlight specific bacterial engineering challenges, particularly beyond HGT concerns. Given the prominence of CRISPR in bacterial research, more critical evaluation of limitations and future enhancements in bacterial genetic engineering would make the comparison more balanced.]

Response 4: [Agree. We have further added more critical evaluation of limitations and future enhancements in bacterial genetic engineering to make the comparison more balanced. More details can be found in our revised manuscript. Thank you!]

 

Comments 5: [Each organism (bacteria, fungi, and oomycetes) faces unique challenges, but the discussion of these challenges feels rushed or incomplete. Provide a deeper examination of the technical limitations—such as off-target effects in fungi, transformation difficulties in oomycetes, or resistance to CRISPR technologies—and offer insights into ongoing research aimed at overcoming these issues.]

Response 5: [Agree. We have further provided a deeper examination of the technical limitations accordingly. More details can be found in our revised manuscript. Thank you!]

 

Comments 6: [There is some mention of ecological impacts and horizontal gene transfer risks, but this discussion could be more prominent throughout. Engage with the broader ethical and ecological debates in biotechnology, including public concern about GMOs, unintended ecological consequences, and long-term risks of deploying genetically modified organisms.]

Response 6: [Agree. We have revised the discussion to make it more prominent throughout. More details can be found in our revised manuscript. Thank you!]

 

Comments 7: [The comparative analysis should draw clearer distinctions between the organisms. Right now, the analysis hints at differences but could be more explicit. Discuss, for example:

 

How bacteria’s single-cell simplicity makes them easier to genetically modify.

How fungi’s multicellular complexity creates barriers to transformation.

How oomycetes’ evolutionary distinctness presents unique challenges in manipulating their genomes.

A table or figure comparing key genetic engineering metrics (e.g., transformation efficiency, off-target risks, ecological concerns) across bacteria, fungi, and oomycetes could help visually reinforce these differences. ]

Response 7: [Agree. We have made the comparative analysis more explicit by further discussing those key points suggested (Table 3). More details can be found in our revised manuscript. Thank you!]

 

Comments 8: [Expand on how specific Cas9 variants or delivery methods (e.g., nanoparticles) will address the existing barriers in each organism. ]

Response 8: [Agree. We have made the expansion accordingly. More details can be found in our revised manuscript. Thank you!]

 

Comments 9: [Explore the future potential of synthetic biology with more detail—what practical applications might emerge from constructing synthetic gene networks or microbial factories? ]

Response 9: [Agree. We have further explored the potential of synthetic biology with more details. More details can be found in our revised manuscript. Thank you!]

 

Comments 10: [Elaborate on the integration of multi-omics approaches (e.g., transcriptomics, proteomics) in discovering new gene targets or improving pathogen resistance, particularly in fungi and oomycetes. ]

Response 10: [Agree. We have further elaborated the integration of multi-omics approaches accordingly. More details can be found in our revised manuscript. Thank you!]

 

Comments 11: [Highlight emerging tools (like CRISPR/Cas12a or base editors) in greater detail for their specific advantages in addressing the genome-editing challenges in fungi and oomycetes. ]

Response 11: [Agree. We have further highlighted emerging tools such as CRISPR/Cas12 and base editors in engineering microbes accordingly. More details can be found in our revised manuscript. Thank you!]

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript reads fine now. I endorse it for publication.