Synthetic Biology-Based Approaches to Investigate Host–Pathogen Interactions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this review manuscript, the author summarizes recent advances in host-pathogen interactions inspired by synthetic biology. Specifically, the paper explores various topics, including CRISPR-based tools, genetic circuits in whole cells, phage therapy approaches, and organ-on-a-chip platforms. These areas highlight the interplay between synthetic biology and host-pathogen interactions, offering unique perspectives for researchers in these fields. Overall, this review provides a comprehensive and systematic overview. However, several points need further attention and revision. I recommend that the paper be considered for publication in SynBio after addressing the following concerns and suggestions.

1. (Inappropriate initial placement of abbreviations) This paper includes numerous abbreviations for specific terms. However, some are not introduced appropriately at their first mention. For instance, “MDR” (multi-drug-resistant) is not defined at its initial appearance in the main text on line 29 but instead is introduced in line 711. The author should carefully review similar cases to improve the paper’s consistency and readability.

2. (Inconsistent formatting of identical terms/phrases) There are inconsistencies in the formatting of the same terms or phrases. For example, “MDR” is defined as “multi-drug-resistant” in line 29 but appears as “multidrug-resistant” in line 35. Additionally, the “sp.” formatting differs between line 87 and line 222. It is recommended that the author thoroughly check for and correct similar inconsistencies.

3. (Non-standard expressions) Some phrases in the manuscript do not follow standard scientific conventions. For example, it is preferable to use “Lambda-Red recombination” instead of “l-Red recombination” in line 43. Similarly, “researchers in synthetic biology” may be more appropriate than “synthetic biologist” in line 127. Revising such terms would enhance the manuscript’s scientific accuracy and clarity.

4. (Typos) The manuscript contains typographical errors, including but not limited to:

Line 46: “Oher”.

Line 554: “Importanntly”.

Figure 3: “Rapture”.

A careful proofreading of the entire manuscript is advised to address these issues.

5. (Necessity of full terms for abbreviations) Some abbreviations lack their full terms upon first mention. For instance, in line 48, “Tn-seq” should be expanded to clarify what “Tn” stands for. Similarly, the full term for “tracr-RNA” should be included at its first appearance in line 183. Addressing these omissions will ensure clarity for readers unfamiliar with these terms.

6. (Detailed explanation) In lines 78-79, it is recommended to provide an explanation of why double-strand breaks cause death in bacterial species but not in eukaryotes. For instance, most prokaryotes lack the non-homologous end joining (NHEJ) system needed to repair these breaks. Including this detail would improve the scientific depth and clarity of the manuscript.

7. (Logical connections in the introduction) The introduction section lacks logical flow between paragraphs, making it unclear how the details presented relate to the main topic or to one another. It is suggested that the author revise this section to strengthen the logical connections and improve the overall readability of the article.

8. (Simplify subtitle 2) Subtitle 2 is overly long and unnecessarily detailed. Simplifying it would make it more concise and reader-friendly.

9. (Incomplete coverage) In line 176, the uses of genome editing are not comprehensively addressed. While bacteria, plants, and mammals are mentioned, other important animal models are omitted. This section should be expanded to include these additional examples.

10. In line 192, the term “These deletion mutants” is inaccurately used to describe CRISPRi-based technology. A more accurate term, such as “These knock-down mutants”, would be appropriate.

11. (Relevance of CRISPRa) The discussion of CRISPRa in lines 245-265 lacks clear relevance to the manuscript’s focus. There is no connection established between CRISPRa and host-pathogen interactions. It is recommended to add relevant examples to justify the inclusion of this section or to remove it entirely.

12. (Figure 1 inconsistencies) In Figure 1, there are inconsistencies in the use of uppercase and lowercase letters, and the use of “-a” in “CRISPR-Cas-a tool” is unclear or misleading. These issues should be addressed for clarity and accuracy.

13. (Figure 2 readability) Some text in Figure 2 is too small to read, especially for labels such as promoters. It is recommended to enlarge these to improve readability.

14. In line 441, IPTG and ATC are incorrectly referred to as inducible promoters. These are inducers, not promoters. This should be revised.

15. (Figure 3 formatting) In Figure 3, there are missing spaces in the phrase “Antimicrobial resistance/ essential/ virulence gene” before each “/”. This should be corrected.

16. In line 581, “Figure 4” should be formatted in bold, consistent with other figure references in the manuscript.

17. (Clarity of Figure 4) In Figure 4, panel B, the term “Strain” is unclear and may confuse readers. It should be explicitly defined or clarified to indicate what it represents.

18. (Adding a schematic diagram) To improve the manuscript’s clarity and organization, it is suggested to add a schematic diagram (potentially as a new Figure 1) illustrating the review’s framework. This diagram could outline the key relations of synthetic biology to four sections: CRISPR, genetic circuits, phage therapy, and organ-on-a-chip. Such a figure would greatly enhance the readability and provide readers with a clear summary of the paper’s structure. While this is an optional suggestion, it would be a valuable addition.

Author Response

Response to reviewer -1: Synbio manuscript id: synbio-3400221

 

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files.

 

Comment-1: (Inappropriate initial placement of abbreviations) This paper includes numerous abbreviations for specific terms. However, some are not introduced appropriately at their first mention. For instance, “MDR” (multi-drug-resistant) is not defined at its initial appearance in the main text on line 29 but instead is introduced inline 711. The author should carefully review similar cases to improve the paper’s consistency and readability.

 

Response-1: Thank you for pointing this out. I agree with the comment. Therefore, I have changed MDR to multi-drug-resistant (MDR) in its first appearance line 8 (Abstract) as well as in line 57 in the introduction section.

 

Comment-2a: (Inconsistent formatting of identical terms/phrases) There are inconsistencies in the formatting of the same terms or phrases. For example, “MDR” is defined as “multi-drug-resistant” in line 29 but appears as “multidrug-resistant” in line 35.

 

Response-2a: I have changed MDR to multi-drug-resistant across the manuscript. These can be found in the following line numbers: 8, 57, 64, 152, 298, 300, 916, 959, 1064, and 1396.

 

Comment- 2b: Additionally, the “sp.” formatting differs between line 87 and line 222. It is recommended that the author thoroughly check for and correct similar inconsistencies.

 

Response 2b: I have the word replace the sp with the italicized “sp” across the manuscript. These can be found in lines 166, 1011, and 1012. The Pseudomonas sp in line 222 of the original submission is replaced with Pseudomonas fluorescens in line 507.

 

 

Commment-3a: (Non-standard expressions) Some phrases in the manuscript do not follow standard scientific conventions. For example, it is preferable to use “Lambda-Red recombination” instead of “l-Red recombination” in line 43.

 

Response 3a: I have replaced “l-Red recombination” with “Lambda-Red recombination” in the line number 73 of the revised manuscript.

 

Comment-3b: Similarly, “researchers in synthetic biology” may be more appropriate than “synthetic biologist” in line 127. Revising such terms would enhance the manuscript’s scientific accuracy and clarity.

 

Response 3b: I have replaced “synthetic biologist” with “researchers in synthetic biology” in line number 269 of the revised manuscript.

 

Comment 4. (Typos) The manuscript contains typographical errors, including but not limited to:

Line 46: “Oher”.

Line 554: “Importanntly”.

Figure 3: “Rapture”.

A careful proofreading of the entire manuscript is advised to address these issues.

 

Response 4; I have replaced

“Other” with “Other” in line 75 of the revised manuscript

“Importanntly” with “Importantly” in line 1081 of the revised manuscript.

Figure 3: Rapture is replaced with” Rupture”.

 

Comment 5. (Necessity of full terms for abbreviations) Some abbreviations lack their full terms upon first mention. For instance, in line 48, “Tn-seq” should be expanded to clarify what “Tn” stands for. Similarly, the full term for “tracr-RNA” should be included at its first appearance in line 183. Addressing these omissions will ensure clarity for readers unfamiliar with these terms.

 

Response 5: I have added the term “Transposon insertion sequencing” before Tn-seq for clarity in line number 78 and the word “trans-activating CRISPR RNA” before tracr-RNA in line number 402 of the revised manuscript for better clarity.

 

Comment 6. (Detailed explanation) In lines 78-79, it is recommended to provide an explanation of why double-strand breaks cause death in bacterial species but not in eukaryotes. For instance, most prokaryotes lack the non-homologous end joining (NHEJ) system needed to repair these breaks. Including this detail would improve the scientific depth and clarity of the manuscript.

 

Response 6: I have added the line “The widely used Type II CRISPR system consists of a chimeric RNA called guide RNA and a single nuclease, Cas9, that can introduce double-stranded breaks in the DNA, a process that is lethal for most bacterial species [28] due to the lack of non-homologous end joining (NHEJ) system needed to repair these breaks” (line 158) for better understanding.

 

Comment-7. (Logical connections in the introduction) The introduction section lacks logical flow between paragraphs, making it unclear how the details presented relate to the main topic or to one another. It is suggested that the author revise this section to strengthen the logical connections and improve the overall readability of the article.

 

Response-7: The introduction section has been rewritten for better clarity. I have added sentences for better flow between the different paragraphs. Some additional lines have been incorporated for better understanding. Please see the revised manuscript where I have used track change to show the changes I made to the initially submitted manuscript.

 

Comment-8: (Simplify subtitle 2) Subtitle 2 is overly long and unnecessarily detailed. Simplifying it would make it more concise and reader-friendly.

 

Response-8: I have modified the Subtitle to “CRISPR- a versatile tool for studying antimicrobial resistance and gene regulation” (line 713).

 

Comment-9:(Incomplete coverage) In line 176, the uses of genome editing are not comprehensively addressed. While bacteria, plants, and mammals are mentioned, other important animal models are omitted. This section should be expanded to include these additional examples.

 

Response -9: I have added the following paragraph for better clarity (line number 377-391):

“For example, CRISPR-Cas technology has enabled researchers to modify plant genomes to enhance desirable traits such as yield, nutritional content, and resistance to diseases and pests. For instance, CRISPR-Cas has been used to develop crops that can withstand abiotic stresses like drought, salinity, and extreme temperatures, which are becoming increasingly important due to climate change [73]. Additionally, it has facilitated the creation of plants with improved herbicide tolerance and reduced reliance on chemical treatments [74]. Finally, CRISPR-Cas technology is paving the way for sustainable agricultural practices and the development of crops that can meet the growing global food demand [75]. CRISPR-Cas technology has revolutionized mammalian genetic research by enabling precise genome editing. Widely used in creating animal models, particularly mice, it facilitates the study of gene functions in disease development and progression [76]. Additionally, CRISPR-Cas is employed in reproductive biology to edit genes in oocytes and early embryos, facilitating studies on developmental processes and genetic disorders [77]. This technology also holds promise for therapeutic applications, such as correcting genetic mutations in somatic cells to treat inherited diseases [78].”

 

Comment-10. In line 192, the term “These deletion mutants” is inaccurately used to describe CRISPRi-based technology. A more accurate term, such as “These knock-down mutants”, would be appropriate.

Response 10: I have replaced “These deletion mutants” with “These knock-down mutants” in line 411 of the revised manuscript.

 

Comment-11. (Relevance of CRISPRa) The discussion of CRISPRa inlines 245-265 lacks clear relevance to the manuscript’s focus. There is no connection established between CRISPRa and host-pathogen interactions. It is recommended to add relevant examples to justify the inclusion of this section or to remove it entirely.

 

Response-11: I have added the following lines for better clarity (lines 597-616)):

“One challenge in studying host-pathogen interactions in bacterial systems is the absence of suitable PAM sites in target genes, complicating the use of CRISPRa for gene regulation. To address this, Kiattisewee et al, systematically evaluated PAM-flexible dCas9 variants for their gene activation potential [97]. The authors found that dxCas9-NG exhibited a high dynamic range at NGN PAM sites, while dSpRY enabled modest activity across nearly all PAMs. However, increased PAM flexibility reduced CRISPR interference (CRISPRi) efficacy, which could be partially rescued by multiplexing sgRNAs. This study offers a valuable framework for optimizing dCas9 variants to expand CRISPRa/i applications in bacterial gene regulation”.

 

Comment-12: (Figure 1 inconsistencies) In Figure 1, there are inconsistencies in the use of uppercase and lowercase letters, andt he use of “-a” in “CRISPR-Cas-a tool” is unclear or misleading. These issues should be addressed for clarity and accuracy.

 

Response-12: Figure 1 is modified by including all the suggestions. Please see the revised Figure 1.

 

Comment-13: (Figure 2 readability) Some text in Figure 2 is too small to read, especially for labels such as promoters. It is recommended to enlarge these to improve readability.

 

Response-13: Figure 2 is modified by including all the suggestions. Please see the revised Figure 2.

 

Comment-14: In line 441, IPTG and ATC are incorrectly referred to as inducible promoters. These are inducers, not promoters. This should be revised.

 

Response-14: I have rewritten line 903 of the revised manuscript as “Sensors and response regulators were driven by either inducible promoters (induced by Isopropyl β-D-1-thiogalactopyranoside, IPTG, or ATC) in vitro or constitutive promoters (J23104, J23105, and J23109) in murine infection models”.

 

 

Comment-15: (Figure 3 formatting) In Figure 3, there are missing spaces in the phrase “Antimicrobial resistance/ essential/ virulence gene “before each “/”. This should be corrected.

 

Response-15: Spaces are added in the phrase “Antimicrobial resistance/ essential/ virulence gene “before each “/” of Figure 3. The modified figure is uploaded.

 

Comment-16: In line 581, “Figure 4” should be formatted in bold, consistent with other figure references in the manuscript.

 

Response-16: Figure 4 is formatted to bold. Please see the revised Figure 4.

 

Comment-17.: (Clarity of Figure 4) In Figure 4, panel B, the term “Strain“ is unclear and may confuse readers. It should be explicitly defined or clarified to indicate what it represents.

 

Response-17: Figure 4 and the associated figure legend is modified. I have added two new panels-Figure 4C and Figure 4D. The figure legend is rewritten as the following:

“Figure 4. Organ-on-a-Chip to study host-pathogen interactions. A. Overview of various organs modeled using organ-on-a-chip technology to investigate the molecular mechanisms of infectious diseases and the potential applications of this approach. Organ on a chip has been used to decipher bacterial and host response, to study the effect of drugs, antimicrobials and engineered bacteriophages (phage therapy) on bacterial pathogens, and to study genetic and epigenetic changes in response to the environment. B. Simplified diagram of a model organ-on-chip illustrating the layered structure of the chip. The top layer contains the pathogenic bacteria of interest, followed by the extracellular matrix. Nutrients, growth factors, and drugs flow through this layer to replicate the host environment (e.g., gut). Beneath this, the first host cell layer is represented, such as umbrella cells in a urinary bladder model. The chip design includes mechanical strain to simulate physiological conditions, such as peristaltic motion in a gut model or the stress bladder epithelial cells undergo during filling and voiding in a urinary bladder model. A porous membrane is located below the host cell layer, enabling efficient exchange of chemicals with the underlying tissue layer. C. Bladder-on-a-Chip: A Tool Mimicking the Urinary Bladder Environment: The bladder-on-a-chip is an innovative device designed to replicate the environment of the urinary bladder. It consists of an upper chamber lined with bladder epithelial cells and a lower chamber containing endothelial cells. By incorporating mechanical strain that simulates the natural expansion of bladder cells during urine storage and their subsequent relaxation during micturition, this device closely mimics the in vivo conditions of the urinary bladder. This realistic model provides a valuable platform for studying host-pathogen interactions. D. Bladder-on-a-Chip in Re-current UTI Studies: The bladder-on-a-chip has been used to investigate the mechanisms underlying recurrent urinary tract infections (rUTIs). Uropathogenic Escherichia coli (UPEC) were introduced into the upper chamber, where they adhered to the epithelial cells. Some UPEC evaded the host immune response by invading the epithelial cells and forming intracellular bacterial communities (IBCs). Upon antibiotic treatment, extracellular UPEC that failed to invade the cells were eliminated, but those within IBCs were shielded from the antibiotics. These protected bacte-ria multiplied within the host cells and were released into the external environment upon host cell rupture. The released UPEC then infected new epithelial cells, perpetuating the cycle of recurrent UTIs”.

 

The word “strain” is explained in detailed in the manuscript by incorporation of this new paragraph (line 1157-line 1172):

 

“The microfluidic chip harbors a layer of bladder cells at the base of a channel filled with diluted human urine. The bladder cells were infected with uropathogenic Escherichia coli (UPEC) and imaged over time to observe bacterial movement, interactions with the bladder cells, and aggregation. Immune cells derived from human blood were introduced into a vascular channel beneath the bladder tissue, lined with endothelial cells mimicking blood vessel architecture. The immune cells swiftly crossed the endothelial barrier into the bladder tissue, targeting infection sites. To mimic the bladder filling due to accumulation of urine and quick voiding due to micturition, the authors designed a duty cycle (periodic application of mechanical or biochemical stimuli to mimic the natural rhythms and cycles experienced by tissues in the body) of 6 hr. The cycle included a linear increase in strain to simulate bladder filling (0–2 hours), sustained stretching to represent a filled bladder (2–4 hours), rapid strain relaxation over 2 minutes to mimic voiding (4:02 hours), and maintenance without strain to reflect a voided bladder (4:02–6 hours).To facilitate the attachment of the pathogen to the epithelial cells, the flow rate of the urine harboring the pathogen was selected to be 1.2ml/hr which corresponded to a shear stress of 0.02 dyne cm−2 for a period of 1.5–2 hr. (Figure 4C)”.

 

 

Comment-18. (Adding a schematic diagram) To improve the manuscript’s clarity and organization, it is suggested to add a schematic diagram (potentially as a new Figure 1) illustrating the review’s framework. This diagram could outline the key relations of synthetic biology to four sections: CRISPR, genetic circuits, phage therapy, and organ-on-a-chip. Such a figure would greatly enhance the readability and provide readers with a clear summary of the paper’s structure. While this is an optional suggestion, it would be a valuable addition.

 

Response-18: The Graphical summary explains the review nicely as it contains all the 4 technologies I am discussing in this review.  Please see the graphical abstract. So, I am not adding any new figure.

 

 

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files.

 

Comment-1: (Inappropriate initial placement of abbreviations) This paper includes numerous abbreviations for specific terms. However, some are not introduced appropriately at their first mention. For instance, “MDR” (multi-drug-resistant) is not defined at its initial appearance in the main text on line 29 but instead is introduced inline 711. The author should carefully review similar cases to improve the paper’s consistency and readability.

 

Response-1: Thank you for pointing this out. I agree with the comment. Therefore, I have changed MDR to multi-drug-resistant (MDR) in its first appearance line 8 (Abstract) as well as in line 57 in the introduction section.

 

Comment-2a: (Inconsistent formatting of identical terms/phrases) There are inconsistencies in the formatting of the same terms or phrases. For example, “MDR” is defined as “multi-drug-resistant” in line 29 but appears as “multidrug-resistant” in line 35.

 

Response-2a: I have changed MDR to multi-drug-resistant across the manuscript. These can be found in the following line numbers: 8, 57, 64, 152, 298, 300, 916, 959, 1064, and 1396.

 

Comment- 2b: Additionally, the “sp.” formatting differs between line 87 and line 222. It is recommended that the author thoroughly check for and correct similar inconsistencies.

 

Response 2b: I have the word replace the sp with the italicized “sp” across the manuscript. These can be found in lines 166, 1011 and 1012. The Pseudomonas sp in the line 222 of the original submission is replace with Pseudomonas fluorescens in the line 507.

 

 

Commment-3a: (Non-standard expressions) Some phrases in the manuscript do not follow standard scientific conventions. For example, it is preferable to use “Lambda-Red recombination” instead of “l-Red recombination” in line 43.

 

Response 3a: I have replaced “l-Red recombination” with “Lambda-Red recombination” in the line number 73 of the revised manuscript.

 

Comment-3b: Similarly, “researchers in synthetic biology” may be more appropriate than “synthetic biologist” in line 127. Revising such terms would enhance the manuscript’s scientific accuracy and clarity.

 

Response 3b: I have replaced “synthetic biologist” with “researchers in synthetic biology” in the line number 269 of the revised manuscript.

 

Comment 4. (Typos) The manuscript contains typographical errors, including but not limited to:

Line 46: “Oher”.

Line 554: “Importanntly”.

Figure 3: “Rapture”.

A careful proofreading of the entire manuscript is advised to address these issues.

 

Response 4; I have replaced

“Other” with “Other” in line 75 of the revised manuscript

“Importanntly” with “Importantly” in line 1081 of the revised manuscript.

Figure 3: Rapture is replaced with” Rupture”.

 

Comment 5. (Necessity of full terms for abbreviations) Some abbreviations lack their full terms upon first mention. For instance, in line 48, “Tn-seq” should be expanded to clarify what “Tn” stands for. Similarly, the full term for “tracr-RNA” should be included at its first appearance in line 183. Addressing these omissions will ensure clarity for readers unfamiliar with these terms.

 

Response 5: I have added term “Transposon insertion sequencing” before Tn-seq for clarity in the line number 78 and the word “trans-activating CRISPR RNA” before tracr-RNA in the line number 402 of the revised manuscript for better clarity.

 

Comment 6. (Detailed explanation) In lines 78-79, it is recommended to provide an explanation of why double-strand breaks cause death in bacterial species but not in eukaryotes. For instance, most prokaryotes lack the non-homologous end joining (NHEJ) system needed to repair these breaks. Including this detail would improve the scientific depth and clarity of the manuscript.

 

Response 6: I have added the line “The widely used Type II CRISPR system consists of a chimeric RNA called guide RNA and a single nuclease, Cas9, that can introduce double-stranded breaks in the DNA, a process that is lethal for most bacterial species [28] due to the lack of non-homologous end joining (NHEJ) system needed to repair these breaks” (line 158) for better understanding.

 

Comment-7. (Logical connections in the introduction) The introduction section lacks logical flow between paragraphs, making it unclear how the details presented relate to the main topic or to one another. It is suggested that the author revise this section to strengthen the logical connections and improve the overall readability of the article.

 

Response-7: The introduction section has been rewritten for better clarity. I have added sentences for better flow between the different paragraphs. Some additional lines have been incorporated for better understanding. Please see the revised manuscript where I have used track change to show the changes I made to the initially submitted manuscript.

 

Comment-8: (Simplify subtitle 2) Subtitle 2 is overly long and unnecessarily detailed. Simplifying it would make it more concise and reader friendly.

 

Response-8: I have modified the Subtitle to “CRISPR- a versatile tool for studying antimicrobial resistance and gene regulation” (line 713).

 

9. (Incomplete coverage) In line 176, the uses of genome editing are not comprehensively addressed. While bacteria, plants, and mammals are mentioned, other important animal models are omitted. This section should be expanded to include these additional examples.

 

Response -9: I have added the following paragraph for better clarity (line number 377-391):

“For example, CRISPR-Cas technology has enabled researchers to modify plant genomes to enhance desirable traits such as yield, nutritional content, and resistance to diseases and pests. For instance, CRISPR-Cas has been used to develop crops that can withstand abiotic stresses like drought, salinity, and extreme temperatures, which are becoming increasingly important due to climate change [73]. Additionally, it has facilitated the creation of plants with improved herbicide tolerance and reduced reliance on chemical treatments [74]. Finally, CRISPR-Cas technology is paving the way for sustainable agricultural practices and the development of crops that can meet the growing global food demand [75]. CRISPR-Cas technology has revolutionized mammalian genetic research by enabling precise genome editing. Widely used in creating animal models, particularly mice, it facilitates the study of gene functions in disease development and progression [76]. Additionally, CRISPR-Cas is employed in reproductive biology to edit genes in oocytes and early embryos, facilitating studies on developmental processes and genetic disorders [77]. This technology also holds promise for therapeutic applications, such as correcting genetic mutations in somatic cells to treat inherited diseases [78].”

 

Comment-10. In line 192, the term “These deletion mutants” is inaccurately used to describe CRISPRi-based technology. A more accurate term, such as “These knock-down mutants”, would be appropriate.

 

Response 10: I have replaced “These deletion mutants” with “These knock-down mutants” in the line number 411 of the revised manuscript.

 

Comment-11. (Relevance of CRISPRa) The discussion of CRISPRa inlines 245-265 lacks clear relevance to the manuscript’s focus. There is no connection established between CRISPRa and host-pathogen interactions. It is recommended to add relevant examples to justify the inclusion of this section or to remove it entirely.

 

Response-11: I have added the following lines for better clarity (line 597-616)):

“One challenge in studying host-pathogen interactions in bacterial systems is the absence of suitable PAM sites in target genes, complicating the use of CRISPRa for gene regulation. To address this, Kiattisewee et al, systematically evaluated PAM-flexible dCas9 variants for their gene activation potential [97]. The authors found that dxCas9-NG exhibited a high dynamic range at NGN PAM sites, while dSpRY enabled modest activity across nearly all PAMs. However, increased PAM flexibility reduced CRISPR interference (CRISPRi) efficacy, which could be partially rescued by multiplexing sgRNAs. This study offers a valuable framework for optimizing dCas9 variants to expand CRISPRa/i applications in bacterial gene regulation”.

 

Comment-12: (Figure 1 inconsistencies) In Figure 1, there are inconsistencies in the use of uppercase and lowercase letters, andt he use of “-a” in “CRISPR-Cas-a tool” is unclear or misleading. These issues should be addressed for clarity and accuracy.

 

Response-12: Figure 1 is modified by including all the suggestions. Please see the revised Figure 1.

 

Comment-13: (Figure 2 readability) Some text in Figure 2 is too small to read, especially for labels such as promoters. It is recommended to enlarge these to improve readability.

 

Response-13: Figure 2 is modified by including all the suggestions. Please see the revised Figure 2.

 

Comment-14: In line 441, IPTG and ATC are incorrectly referred to as inducible promoters. These are inducers, not promoters. This should be revised.

 

Response-14: I have rewritten the line 903 of the revised manuscript as “Sensors and response regulators were driven by either inducible promoters (induced by Isopropyl β-D-1-thiogalactopyranoside, IPTG, or ATC) in vitro or constitutive promoters (J23104, J23105, and J23109) in murine infection models”.

 

 

Comment-15: (Figure 3 formatting) In Figure 3, there are missing spaces in the phrase “Antimicrobial resistance/ essential/ virulence gene “before each “/”. This should be corrected.

 

Response-15: Spaces are added in the phrase “Antimicrobial resistance/ essential/ virulence gene “before each “/” of Figure 3. The modified figure is uploaded.

 

Comment-16: In line 581, “Figure 4” should be formatted in bold, consistent with other figure references in the manuscript.

 

Response-16: Figure 4 is formatted to bold. Please see the revised Figure 4.

 

Comment-17.: (Clarity of Figure 4) In Figure 4, panel B, the term “Strain“ is unclear and may confuse readers. It should be explicitly defined or clarified to indicate what it represents.

 

Response-17: Figure 4 and the associated figure legend is modified. I have added two new panels-Figure 4C and Figure 4D. The figure legend is rewritten as following

“Figure 4. Organ-on-a-Chip to study host-pathogen interactions. A. Overview of various organs modeled using organ-on-a-chip technology to investigate the molecular mechanisms of infectious diseases and the potential applications of this approach. Organ on a chip has been used to decipher bacterial and host response, to study the effect of drugs, antimicrobials and engineered bacteriophages (phage therapy) on bacterial pathogens, and to study genetic and epigenetic changes in response to the environment. B. Simplified diagram of a model organ-on-chip illustrating the layered structure of the chip. The top layer contains the pathogenic bacteria of interest, followed by the extracellular matrix. Nutrients, growth factors, and drugs flow through this layer to rep-licate the host environment (e.g., gut). Beneath this, the first host cell layer is represented, such as umbrella cells in a urinary bladder model. The chip design includes mechanical strain to simulate physiological conditions, such as peristaltic motion in a gut model or the stress bladder epithelial cells undergo during filling and voiding in a urinary bladder model. A porous membrane is located below the host cell layer, enabling efficient exchange of chemicals with the underlying tissue layer. C. Bladder-on-a-Chip: A Tool Mimicking the Urinary Bladder Environment: The bladder-on-a-chip is an innovative device designed to replicate the environment of the urinary bladder. It consists of an upper chamber lined with bladder epithelial cells and a lower chamber containing endothelial cells. By incorporating mechanical strain that simulates the natural ex-pansion of bladder cells during urine storage and their subsequent relaxation during micturition, this device closely mimics the in vivo conditions of the urinary bladder. This realistic model pro-vides a valuable platform for studying host-pathogen interactions. D. Bladder-on-a-Chip in Re-current UTI Studies: The bladder-on-a-chip has been used to investigate the mechanisms under-lying recurrent urinary tract infections (rUTIs). Uropathogenic Escherichia coli (UPEC) were introduced into the upper chamber, where they adhered to the epithelial cells. Some UPEC evaded the host immune response by invading the epithelial cells and forming intracellular bacterial communities (IBCs). Upon antibiotic treatment, extracellular UPEC that failed to invade the cells were eliminated, but those within IBCs were shielded from the antibiotics. These protected bacte-ria multiplied within the host cells and were released into the external environment upon host cell rupture. The released UPEC then infected new epithelial cells, perpetuating the cycle of recurrent UTIs”.

 

The word “strain” is explained in detailed in the manuscript by incorporation of this new paragraph (line 1157-line 1172):

 

“The microfluidic chip harbors a layer of bladder cells at the base of a channel filled with diluted human urine. The bladder cells were infected with uropathogenic Escherichia coli (UPEC) and imaged over time to observe bacterial movement, interactions with the bladder cells, and aggregation. Immune cells derived from human blood were introduced into a vascular channel beneath the bladder tissue, lined with endothelial cells mimicking blood vessel architecture. The immune cells swiftly crossed the endothelial barrier into the bladder tissue, targeting infection sites. To mimic the bladder filling due to accumulation of urine and quick voiding due to micturition, the authors designed a duty cycle (periodic application of mechanical or biochemical stimuli to mimic the natural rhythms and cycles experienced by tissues in the body) of 6 hr. The cycle included a linear increase in strain to simulate bladder filling (0–2 hours), sustained stretching to represent a filled bladder (2–4 hours), rapid strain relaxation over 2 minutes to mimic voiding (4:02 hours), and maintenance without strain to reflect a voided bladder (4:02–6 hours).To facilitate the attachment of the pathogen to the epithelial cells, the flow rate of the urine harboring the pathogen was selected to be 1.2ml/hr which corresponded to a shear stress of 0.02 dyne cm−2 for a period of 1.5–2 hr. (Figure 4C)”.

 

 

Comment-18. (Adding a schematic diagram) To improve the manuscript’s clarity and organization, it is suggested to add a schematic diagram (potentially as a new Figure 1) illustrating the review’s framework. This diagram could outline the key relations of synthetic biology to four sections: CRISPR, genetic circuits, phage therapy, and organ-on-a-chip. Such a figure would greatly enhance the readability and provide readers with a clear summary of the paper’s structure. While this is an optional suggestion, it would be a valuable addition.

 

Response-18: The Graphical summary explains the review nicely as it contains all the 4 technologies I am discussing in this review.  Please see the graphical abstract. So, I am not adding any new figure.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Organ-on-a-chip (OoC) technology is a revolutionary platform that enables the replication of human organ-level functions in vitro, providing an innovative solution to study complex biological processes such as host-pathogen interactions. Traditional models, such as animal models, often fall short due to limitations in human translatability, ethical concerns, and the inability to replicate the dynamic environment of human tissues. The advent of OoC models addresses these challenges by offering a more physiologically relevant and ethically acceptable alternative, enabling real-time studies of pathogen behavior within human organ systems. This review examines the application of OoC technology in investigating host-pathogen interactions, emphasizing the unique advantages it offers over traditional models. We explore how OoC models have been employed to study the dynamic interplay between pathogens and various human organs, including the gut, lungs, and urinary bladder, as well as their role in deciphering host immune responses and microbial dynamics. Additionally, we delve into how OoC technology facilitates the study of host-microbiota interactions, an emerging area of research with profound implications for human health. This review highlights the latest advancements, ongoing challenges, and future directions in the use of OoC models for studying infectious diseases, providing a comprehensive overview of the current state of the field. However, there are some concerns that need to be addressed before processing further:

Title:

• "Synthetic biology-based approaches to probe host-pathogen interactions"
• "Probe" is a weak verb choice in the context of scientific research. A more precise term like "investigate," "analyze," or "understand" would be more appropriate.

Abstract:

• The abstract could benefit from a clearer central thesis statement. While it mentions synthetic biology’s potential, it could highlight the specific contributions made by the authors and how these technologies could change host-pathogen interaction studies. For example, the introduction could better clarify what novel synthetic biology tools have been implemented, beyond CRISPR and engineered circuits.
• "This review explores the potential of synthetic biology as a promising tool for investigating host-pathogen interactions and offering alternative therapeutic solutions..."
• The phrase "alternative therapeutic solutions" is somewhat vague. Instead, specify that synthetic biology might help in designing new diagnostic tools or targeted therapies, especially in the context of MDR pathogens.

• "Bacterial infections are a worldwide threat..."consider adding recent statistics or examples from public health databases to emphasize the urgency of the situation, especially concerning multi-drug resistance (MDR).
• The text states "nosocomial acquired pathogen", which should be "nosocomially acquired pathogen" for proper grammar.
• The review touches on several cutting-edge approaches in synthetic biology, but the overall integration of how these technologies might work together to provide a holistic solution could be more explicit. For example, the use of synthetic biology tools like engineered bacteria in diagnostics and phage therapy should be framed as part of a comprehensive strategy to combat MDR pathogens rather than isolated technologies.

• The review mentions Tn-seq and CRISPRi as important methods for investigating gene function, but fails to provide enough critical evaluation of their limitations. For example, while the paper discusses some drawbacks of Tn-seq, it could also highlight the limitations of CRISPR-based methods in terms of off-target effects or the difficulty in using them across various species, which could hinder their application in diverse host-pathogen models.
• There is also a missed opportunity to discuss the complexity of host-pathogen interactions. Synthetic biology-based systems such as CRISPR and phage therapy are promising, but it would be beneficial to expand on how these technologies account for the multifaceted nature of these interactions. For example, many pathogens interact with multiple hosts and influence a variety of signaling pathways within a host. A brief mention of how synthetic biology can model or simplify these complex interactions could be impactful.

"CRISPR- a versatile tool for studying antimicrobial resistance and gene regulation and bacterial virulence"

• The phrase “Bacteria have by intrinsic restriction systems...” seems grammatically incorrect. It should be “Bacteria have intrinsic restriction systems...”
• "The DNA-targeting activity of the CRISPR-Cas system was demonstrated in the pathogen Staphylococcus epidermidis" is generally correct, but it should specify the nature of the demonstration (e.g., how CRISPR-Cas system was demonstrated—through mutation, inhibition, etc.).
• "Cas9 in turn cleaved the target DNA by causing double strand breaks (endonucleolytic cleavage)." The verb “cleaved” is correct but could be clearer if rephrased: “Cas9 cleaves the target DNA, causing double-strand breaks.”
• "One of the unique features of the CRISPR system is its inherent flexibility in DNA recognition." This is correct, but it would benefit from a clearer explanation of how this flexibility is achieved (e.g., by modifying the gRNA spacer sequence).
• "CRISPR has been widely used for genome editing in bacteria, plants, and mammals." While correct, it could add more emphasis on the impact of these edits or specific applications.
• The description of the CRISPR-Cas9 system could benefit from more specific context. For instance, why is the Type II CRISPR system preferred? It would be useful to add some additional explanation here about the simplicity and effectiveness of Cas9.
• The term "CRISPR-interference (CRISPRi)" is introduced, but the definition of CRISPRi could be clearer for readers unfamiliar with the topic. For example, a brief mention of how CRISPRi differs from CRISPR-Cas9 could be helpful.
• The description of the CRISPRi library screening in Mycobacterium tuberculosis is scientifically robust, but it could benefit from a clearer explanation of how the identified genes influence resistance to the antitubercular drug bedaquiline (BDQ). More detailed data would add clarity.
• The study on Legionella pneumophila is compelling but would benefit from more details about how the engineered CRISPR-based system worked in practical terms. For example, it could be helpful to explain how the simultaneous silencing of 10 genes was validated, and what precise impacts this had on virulence.
• The section on biofilm formation is generally good, but it should clarify the relationship between CRISPRi and biofilm inhibition in more detail. Does the inhibition of these genes reduce biofilm formation directly, or are there other mechanisms at play?
• When discussing essential gene knockouts using mismatches in the spacer sequence of gRNAs, it would be helpful to briefly explain how this fine-tuning process works and why it is particularly important for essential genes, which cannot be completely knocked out without causing lethality. Providing examples from recent literature would strengthen this point.
• The study on Bacillus subtilis and its antibiotic interaction with MAC-0170636 is an interesting application, but it lacks specific conclusions. For example, how does the targeting of the enzyme undecaprenyl pyrophosphate synthase (UppS) provide new therapeutic options?

"Engineered microbes-modern biosensors for pathogen detection and elimination"

• Consider rewording the title to reduce ambiguity. For example, "Engineered Microbes as Synthetic Biosensors for Detection and Elimination of Pathogens".
• The opening line is relatively clear, but a more formal and specific explanation of the terms might be beneficial, especially since readers may vary in their familiarity with synthetic biology. Mentioning the importance of pathogen detection and the role of synthetic biology in medical and environmental contexts early on would add context.
• The discussion of engineered bacteria, such as E. coli and E. coli Nissle, is generally correct, but more precision is needed in the descriptions of the synthetic circuits. For example, the mention of "bistable lambda cI/Cro switch" (lines 336-337) could benefit from a clearer explanation of the biological mechanism, particularly for readers unfamiliar with genetic memory systems.
• The sentence describing the genetic memory system in E. coli (lines 336–344) might be a bit overwhelming for non-specialists. A simpler breakdown of how bistable switches work could help clarify the process. Moreover, the inclusion of "trigger" and "memory" elements could benefit from a brief elaboration on their biological significance and application in diagnostics and therapeutics.
• The flow between the biosensor (3.1) and pathogen-killing (3.2) sections could be more cohesive. Currently, the transition feels abrupt, especially since these topics may overlap more than suggested. Integrating discussions on how biosensors could be paired with therapeutic applications might help bridge the two sections.
• In some parts, technical terminology, such as "type IV secretion system FlgM" (line 420), is used without sufficient context. For a more accessible read, it might help to briefly explain the function of this secretion system and how it contributes to the therapeutic goals. This is especially true for general readers who may not be familiar with bacterial secretion mechanisms.
• The phrase "activatesthe expression" (line 415) should be corrected to "activates the expression".
• In line 419, "secretion module utilizes the type IV secretion system FlgM to release CoPy into the environment" is missing some punctuation between "system" and "FlgM" and could be rephrased for clarity. A better version could be "the secretion module utilizes the type IV secretion system (FlgM) to release CoPy into the environment."

"Phage Therapy-based approaches to decipher host-pathogen interactions"

• Some sections, particularly between the subsections, lack smooth transitions. For instance, the jump from CRISPRi-based phage therapy to endolysin-based approaches is somewhat abrupt. A bridging sentence or summary of how these topics interconnect would help maintain coherence.
• The term "CRISPRi" is used in some places but not always defined or explained sufficiently for readers unfamiliar with the concept. It could be helpful to briefly explain it upon first use.
• Some studies are described in high detail, but the broader relevance of these findings could be explained more clearly. For instance, the Galleria mellonella study is well detailed, but the broader implications of this research could be highlighted in terms of translational potential to human therapy.

Application of CRISPRi-based methods on phage therapy

• The concept of CRISPRi and its application to phage therapy is introduced multiple times without adding new information each time. This could be streamlined by focusing on one example and elaborating on its broader implications.
• For example, references to the work of Citorik et al. are abundant but the discussion could integrate this study better with general trends or challenges in the field, rather than just describing it in isolation.
• "the later often cause the loss" → "the latter often causes the loss" (corrected "later" to "latter" and verb agreement issue with "cause").
• "targeting antibiotic resistance genes blaNDM-1 and blaSHV-18" → "blaNDM-1 and blaSHV-18 antibiotic resistance genes" for clearer phrasing.
• "inducing double-stranded breaks that led to plasmid loss and bacterial death (Figure. 3A)" → "inducing double-stranded breaks that led to plasmid loss and bacterial death (Figure 3A)" (remove unnecessary period before "3A").

Phage-based antimicrobials-endolysins

• The description of endolysins is comprehensive but assumes familiarity with certain biochemical and structural concepts. A bit more background information or simpler phrasing could make it more accessible. For example, it would be useful to explain terms like "modular structure" and "cell wall-binding domain (CBD)" in simpler terms for non-expert readers.
• Some sections are overly technical and could benefit from a clearer summary of the main points. For example, the comparison between Gram-positive and Gram-negative targeting endolysins could be better structured to show the significance of these differences.
• "Endolysin that target Gram-positive bacteria" → "Endolysins that target Gram-positive bacteria" (plural consistency with subject).
• "Inspite" → "In spite" (correct spelling).

Application of engineered phages for bacterial detection and diagnostics

• While the section describes various applications of engineered phages in diagnostics, it lacks a clear conclusion or summary of the importance of these techniques in the broader context of phage therapy. The reader is left with a series of examples but no overarching perspective on their potential or limitations.
• Some sentences in this section could be rewritten to reduce redundancy and improve flow. For example, "This system emitted a luminescent signal when encountering the pathogen, enabling detection..." could be simplified or combined with related sentences to avoid repetition.
• "phage protein through homolo- gous recombination" → "phage protein through homologous recombination" (fix hyphenation and typo).
• "later" → "latter"
• "cause" → "causes"
• "Inspite" → "In spite"
• "target" → "targeting" (for subject-verb agreement)

• "simutaneously" should be "simultaneously".
• "Mutiple" should be "Multiple".
• "cannnot" should be "cannot".

"Organ-on-a-chip: An Emerging Platform for Investigating Host-Pathogen Interactions"

·       While the manuscript mentions the potential of OOC technology in modeling host-pathogen interactions, it often lacks detailed explanations of the biological and mechanical principles behind these models. For example, in section 5.1, when discussing the urinary bladder model, the explanation of the mechanical strain and its physiological relevance could benefit from further elaboration. How does shear stress influence pathogen behavior? A more in-depth exploration of the mechanistic factors influencing pathogen interactions at the cellular level would provide readers with a clearer understanding of the added value of OOC models.

·       In several instances, OOC models are mentioned but without a clear justification of why these specific models were chosen over others. For instance, when describing the Mycobacterium tuberculosis study in the lung-on-a-chip (lines 609-611), there could be a deeper comparison between OOC models and alternative approaches such as ex vivo lung tissue culture or 3D lung organoids. Understanding the advantages of OOC systems—such as better recapitulation of in vivo conditions—over these models could strengthen the argument for their use in studying host-pathogen interactions.

• "Inaccessibility" (line 572): This is a proper term, but the phrase "the inaccessibility of human organs" can be rephrased as "the challenges in accessing human organs" to sound more polished.
• "treament" (line 586): The word should be "treatment."
• "the the host immune" (line 601): This repetition should be corrected to "the host immune."
• "exhihibited" (line 616): The correct spelling is "exhibited."

·       The transition between different pathogens and systems (e.g., UPEC to Mycobacterium tuberculosis to Pseudomonas aeruginosa) can be somewhat abrupt. A brief bridging sentence or paragraph connecting these examples would improve the flow and coherence of the manuscript. Each section is well written, but the overall organization could benefit from stronger cohesion.

·       While the audience may be familiar with terms like "shear stress," "peristaltic motion," and "microvascular epithelial cells," there are moments where these terms are used without sufficient context for readers who may not be experts in OOC technology. A more gradual introduction to these terms with brief explanations or references could improve accessibility.

·       The manuscript frequently mentions figures (e.g., "Figure 4A", "Figure 4B") but does not provide enough context for what the figures are showing. It would be beneficial to add a brief description of each figure's content in the text to guide readers in interpreting the data. The figures themselves should be well-detailed and include scale bars, captions, and clear labeling of components for better understanding.

·       While the studies cited are significant, the manuscript lacks detailed statistical or experimental results from the referenced studies. For example, when discussing the efficacy of OOC systems in replicating in vivo conditions (e.g., Mycobacterium tuberculosis infection in lung-on-a-chip models), providing quantitative data (such as infection rates, cellular responses, or pathogen load changes) could give more weight to the claims.

• Several statements are made about pathogen behavior in OOC models (e.g., "Uropathogenic Escherichia coli evade host immune defenses..."). It would be beneficial to cite specific experimental studies that provide data or experimental protocols used to measure these behaviors in OOC systems.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please see the attachment

Author Response File: Author Response.pdf