Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells

Chao Li; Zhong Liu; Justin Anderson; Zhongyu Liu; Liping Tang; Yao Li; Ning Peng; Jianguo Chen; Xueming Liu; Lianwu Fu; Tim M. Townes; Steven M. Rowe; David M. Bedwell; Jennifer Guimbellot; Rui Zhao

doi:10.1371/journal.pone.0295009

Peer Review History

Original SubmissionJuly 7, 2023
4 Sep 2023 Decision Letter - Alessio Branchini, Editor PONE-D-23-20582 Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells PLOS ONE Dear Dr. Zhao, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Oct 19 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594. NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Competing Interests section: "I have read the journal's policy and the authors of this manuscript have the following competing interests: S.M.R. provided consulting services and received grants from Novartis, TranslateBio, Galapagos/Abbvie, Synedgen/Synspira, Eloxx, Vertex Pharmaceuticals, Ionis, Astra Zenica, Renovion, Cystetic Medicines, and Arcturus. S.M.R. is the inventor or co-inventor of several patents and held stock or stock options of Synedgen/Synspira and Renovion. " Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The study “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, by Li and colleagues is of high quality and of interest to a broad audience. The results presented in the main figures of the manuscript provide a concise and impactful study on the use of prime editing to “fix” CFTR mutation p.W1282X. The major advances presented here are the use of an “all-in-one” helper dependent Adenovirus (HDAd) to efficiently deliver all of the prime editing machinery to iPSC-derived airway epithelial cells and restore p.W1282X-CFTR function. Because of the size of the payload, this has previously not been able to be achieved. This reviewer found much of the interesting biology to be in the supplemental figures, where a good amount of optimization was performed. There are only a few comments to address. Comments: 1) The authors touted the ability of delivering all of the machinery in a helper dependent adenovirus (HDAd) system for efficient editing. However, even with a plasmid system containing all of the PE machinery and high transfection efficiency, the editing efficiency was only 0.36% in iPSCs. (Fig 1D). With the HDAd system providing ~70% transduction efficiency of differentiated airway epithelial cells only corrected 2-3% of the p.W1282X alleles. Indeed, the bottleneck for correction of p.W1282X CF mutations is the overall efficiency of prime editing, not delivery. While there is nice discussion on this issue, the technical limitations of prime editing will take time to resolve. Maybe this bottleneck would not be a significant issue if in that population of transduced cells, basal cells were effectively edited. This would lead to a repopulation of airway epithelial cells with edited alleles and rescued CFTR function, thereby amplifying the therapeutic impact of low-efficiency prime editing. Important for this study, what cell types in the differentiated airway epithelia were transduced by HDAd? Are basal cells included in this population? 2) Why was functional rescue of p.W1282X-CFTR prime edited differentiated epithelial cells not reported? It would seem that even with as low as 1.5% editing there would be measurable rescue of CFTR function by Ussing chamber assays for measurement of short circuit current (Isc). Indeed, the CFTR functional rescue is often significantly higher than the genome editing efficiency. Reviewer #2: I have reviewed the manuscript numbered PONE-D-23-20582, titled “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, submitted by Chao L., Zhong L., et al. This article, presenting an original research, is very interesting and very clearly described. In the manuscript, the authors have developed a novel PRIME editing system for the correction of the W1282X CFTR mutation. The authors did a very good initial screening job for the components of the PRIME editing System (as reported also in supplementary information) and then, validated the best one in iPSCs generated from nasal epithelial cells of a homozygous W1282X cystic fibrosis patient. PE-iPSCs were also differentiated into airway epithelial cells and lung organoids, for testing the restored CFTR function. As a very important point to become a therapeutic modality, the Authors tested the PRIME editing system directly in differentiated airway epithelial cells, using an HDAd vector containing all the sequences needed for the prime editing system (all-in –one vector). In conclusion, the Authors demonstrated the correction of the point mutation albeit in a limited number (2.5-5%) of iPS-derived epithelial cells and, very importantly, getting no indels effect though the DNA. These results could be very useful for the development of an in-vivo PRIME editing system for stop mutation in CF. The only suggestion to the Authors could be to add a sentence in the discussion, in particular in the paragraph stating the editing efficiency, indicating (if it is known) what could hypothetically be the correction rate to be reached by PRIME-CRISPR/Cas9-based systems, in order to get a therapeutic level of CFTR for CF patients. Other specific comments: -Are Results and Conclusions presented in an appropriate fashion and are supported by the data? YES. Results are very well written, linear, and complete. The same is for the Conclusions (see my only suggestion above). -Is the article presented in an intelligible fashion and written in standard English? YES - Does the research meet all applicable standards for the ethics of experimentation and research integrity? YES ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0295009.r001
Revision 1
18 Oct 2023 Author Response We sincerely thank the editor’s comments and feedback. We have updated and formatted the revised manuscript accordingly. Below are our point-to-point responses. Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf � We formatted the revised manuscript according to the style requirements. This includes changing level 1 and 2 headings to appropriate fonts, abiding by the requirements for figure citations/captions, and updating the style of supporting information citations and acknowledgments. We also updated the title, author, and affiliation style to abide by the guidelines. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594." We note that you have provided funding information that is not currently declared in your Funding Statement. � We have updated the funding information in the revised submission. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594. � Funding information has been deleted from the Acknowledgements section. NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. � The below statement has been included in the cover letter. “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 3. Thank you for stating the following in the Competing Interests section: "I have read the journal's policy and the authors of this manuscript have the following competing interests: S.M.R. provided consulting services and received grants from Novartis, TranslateBio, Galapagos/Abbvie, Synedgen/Synspira, Eloxx, Vertex Pharmaceuticals, Ionis, Astra Zenica, Renovion, Cystetic Medicines, and Arcturus. S.M.R. is the inventor or co-inventor of several patents and held stock or stock options of Synedgen/Synspira and Renovion. " Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. � The below statement has been included in the cover letter and revised manuscript. “This does not alter our adherence to PLOS ONE policies on sharing data and materials. " 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. � “data not shown” has been removed from the revised manuscript because it is not a core part of the reported study. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. � Captions for Supporting Information have been added to the end of the revised manuscript to meet the guidelines. [Note: HTML markup is below. Please do not edit.] We sincerely thank the reviewers for their positive comments and critical feedback, which we have used to revise and strengthen our manuscript. Please find the below point-to-point responses. We anticipate that our study on the correction of CFTR W1282X mutation by prime editing will be of great interest to readers of PLOS ONE. Reviewer #1: The study “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, by Li and colleagues is of high quality and of interest to a broad audience. The results presented in the main figures of the manuscript provide a concise and impactful study on the use of prime editing to “fix” CFTR mutation p.W1282X. The major advances presented here are the use of an “all-in-one” helper dependent Adenovirus (HDAd) to efficiently deliver all of the prime editing machinery to iPSC-derived airway epithelial cells and restore p.W1282X-CFTR function. Because of the size of the payload, this has previously not been able to be achieved. This reviewer found much of the interesting biology to be in the supplemental figures, where a good amount of optimization was performed. There are only a few comments to address. Comments: 1) The authors touted the ability of delivering all of the machinery in a helper dependent adenovirus (HDAd) system for efficient editing. However, even with a plasmid system containing all of the PE machinery and high transfection efficiency, the editing efficiency was only 0.36% in iPSCs. (Fig 1D). With the HDAd system providing ~70% transduction efficiency of differentiated airway epithelial cells only corrected 2-3% of the p.W1282X alleles. Indeed, the bottleneck for correction of p.W1282X CF mutations is the overall efficiency of prime editing, not delivery. While there is nice discussion on this issue, the technical limitations of prime editing will take time to resolve. Maybe this bottleneck would not be a significant issue if in that population of transduced cells, basal cells were effectively edited. This would lead to a repopulation of airway epithelial cells with edited alleles and rescued CFTR function, thereby amplifying the therapeutic impact of low-efficiency prime editing. Important for this study, what cell types in the differentiated airway epithelia were transduced by HDAd? Are basal cells included in this population? � We agree that the observed low correction efficiency could hold therapeutic significance if the corrected cells are predominantly basal cells. For iPSC differentiation into airway epithelium, we primarily followed the published protocol (ref. 29 and 30), which demonstrated the presence of basal cells in iPSC-derived epithelium. HDAd can effectively transduce airway basal cells in animal models (ref. 22). However, determining prime editing-corrected cell types is technically challenging. Our current experimental system detects the prime editing-mediated single nucleotide substitution solely by ddPCR. The ddPCR procedure prevents simultaneous cell type identification. A potential solution to this important question is to construct a W1282X reporter iPSC line, in which a GFP reporter will be fused in-frame to the 3’ end of the mutant CFTR W1282X open reading frame. Because of the W1282X mutation, uncorrected airway cells would remain GFP negative but airway cells with correction would become GFP positive. The GFP-positive cells can then be FACS-sorted for marker staining and single-cell sequencing analysis. However, this approach involves a significant amount of efforts and we believe it should become a follow-up study of the current report. We agree this is an important point that should be further explored. We have included a discussion on the potential implications of correcting basal cells in the revised manuscript (Page 15). 2) Why was functional rescue of p.W1282X-CFTR prime edited differentiated epithelial cells not reported? It would seem that even with as low as 1.5% editing there would be measurable rescue of CFTR function by Ussing chamber assays for measurement of short circuit current (Isc). Indeed, the CFTR functional rescue is often significantly higher than the genome editing efficiency. � We agree with the reviewer and have conducted the recommended experiment (supplemental Figure S4). In the prime-edited airway epithelium, we detected a minor Isc spike following FSK stimulation, a response absent in the unedited controls. When compared to the PE-iPSC-derived airway epithelium (Fig. 3B), the amplitude of the FSK-induced Isc is approximately 20-fold lower, which is in line with the expected functional CFTR allele frequencies (~2.5% vs. 50%). However, we did not observe a VX-770-induced Isc change, which could be attributed to the generally low Isc signals of the HDAd-corrected epithelium or other unidentified factors. We have included these new data in the revised manuscript (Fig. S4 and page 13). Reviewer #2: I have reviewed the manuscript numbered PONE-D-23-20582, titled “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, submitted by Chao L., Zhong L., et al. This article, presenting an original research, is very interesting and very clearly described. In the manuscript, the authors have developed a novel PRIME editing system for the correction of the W1282X CFTR mutation. The authors did a very good initial screening job for the components of the PRIME editing System (as reported also in supplementary information) and then, validated the best one in iPSCs generated from nasal epithelial cells of a homozygous W1282X cystic fibrosis patient. PE-iPSCs were also differentiated into airway epithelial cells and lung organoids, for testing the restored CFTR function. As a very important point to become a therapeutic modality, the Authors tested the PRIME editing system directly in differentiated airway epithelial cells, using an HDAd vector containing all the sequences needed for the prime editing system (all-in –one vector). In conclusion, the Authors demonstrated the correction of the point mutation albeit in a limited number (2.5-5%) of iPS-derived epithelial cells and, very importantly, getting no indels effect though the DNA. These results could be very useful for the development of an in-vivo PRIME editing system for stop mutation in CF. The only suggestion to the Authors could be to add a sentence in the discussion, in particular in the paragraph stating the editing efficiency, indicating (if it is known) what could hypothetically be the correction rate to be reached by PRIME-CRISPR/Cas9-based systems, in order to get a therapeutic level of CFTR for CF patients. � We agree that this is an important point. It is believed that restoration of 10 to 35% of CFTR activities would be required to mitigate pulmonary morbidity (PMID: 15510065). Based on this estimation, a correction level ranging from 10 to 35% mutant alleles would be necessary. However, if the corrected cells primarily comprise basal cells, therapeutic advantages could potentially be attained with lower editing efficiency. We have included this discussion in the revised manuscript (page 15). Attachments Attachment Submitted filename: Rebuttal_03.pdf https://doi.org/10.1371/journal.pone.0295009.r002
14 Nov 2023 Decision Letter - Alessio Branchini, Editor Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells PONE-D-23-20582R1 Dear Dr. Zhao, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Alessio Branchini Academic Editor PLOS ONE Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: John D. Lueck ******** https://doi.org/10.1371/journal.pone.0295009.r003
Formally Accepted
17 Nov 2023 Acceptance Letter - Alessio Branchini, Editor PONE-D-23-20582R1 Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells Dear Dr. Zhao: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Alessio Branchini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0295009.r004

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