Peer Review History
Original SubmissionApril 2, 2021 |
---|
PONE-D-21-10870 CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutant embryos PLOS ONE Dear Dr. Mitchell, 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. The study is well planned. Authors have to provide more detailed methodology and also validation of the successful knockouts, which are very much critical for this kind of manuscripts. Please submit your revised manuscript by Jun 24 2021 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. Please include the following items when submitting your revised manuscript:
If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Rajakumar Anbazhagan, Ph. D. Academic Editor PLOS ONE 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 2.PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Additional Editor Comments: Authors have to provide more detailed methodology and also validation of the successful knockouts. [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: Partly Reviewer #2: Partly Reviewer #3: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No Reviewer #3: Yes ********** 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 Reviewer #3: 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: No Reviewer #3: 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 manuscript by Mitchell et al. explore the potential application of CRISPR/Cas9 targeting in Amphiprion occellaris (anemonefish). The authors demonstrate the successful targeting of rhodopsin-like 2B opsin encoding gene (RH2B), involved in vison and Tyrosinase encoding gene (tyr), involved in the production of melanin. They show clear loss of function in tyr mutant embryos and associated phenotype. Based on this the authors suggest that CRISPR/Cas9 tool can be used for gene-editing in anemonefish, thereby it would help in studying gene function. Overall its straightforward methodology study, suggesting the use of CRISPR/Cas9 application in studying gene function in anemonefish. However, the study lacks functional and behavioral studies of mutant anemonefish fish. Further, CRISPR/Cas9 system has already been used in several fish models, such as zebrafish, tilapia, and medaka (27130213, 24728957) to study gene function or to target a specific gene. As it stands—the authors should include the functional and behavioral study of mutant fish otherwise it just an adaption of the protocol in Amphiprion occellaris and offer no technical advancement over already published studies or provide new insight. a. The authors suggest that the mortality of experimental eggs dependent on the injection process rather than sgRNA/Cas9 cytotoxicity, however, this hypothesis is not validated. b. It’s unclear from the study if the off-target analysis is performed. c. The authors should verify germline transmission mutant embryo as it's important to investigate long-term function study. d. The titration of sgRNA/Cas9 doses should be performed to optimize cas9 cutting activity. e. The authors should perform a behavioral and functional study of mutant Amphiprion occellaris to assess the long-term effect of mutation on the viability of the fish. Reviewer #2: Reviewer Comments to Author(s): The manuscript describes the use of CRISPR/Cas9 for genome editing in reef fish or anemonefish. While CRISPR/Cas9 mediated mutagenesis has been performed in a previous publication (1), the rational is that no protocol has detailed how to inject anemonefish to generate mosaic mutants or complete knockouts in the F0 generation. In the manuscript (PONE-D-21-10870), the authors established a system for implementing CRISPR/Cas9 to produce genome edited reef fish. Although the authors microinjection technique affected the survivability of injected embryos, the overall success rate of CRISPR/Cas9 mediated mutagenesis was measurable. Heritability of CRISPR/Cas9 induced mutations were not detected in the injected reef fish due to the model systems long generation time constraints. However, the authors generate mosaic founder (F0) embryos that closely resemble true null mutations, at least in the case of tyr. The ability to generate F0 mutants will allow testing the functional importance of genes, although off-target effect is more common with this approach. The research presented provides the groundwork for CRISPR/Cas9 application in anemonefish. The methods are appropriate, and the results are clear with exceptions. An overarching concern is the claim that efficient knockdown is achieved to generate a complete knockout in the F0 without clearly explaining the mutational analysis. Overall, the manuscript readability can be revised to improve the flow of the text, but there is sufficient information presented for readers to follow the rationale, procedures and glean insights into the application of the CRISPR/Cas9 system for use on reef fish and other model organisms with longer generation times. Injected embryos are often referred to as founders or F0 and not G0. Lines 32, 36, 103 – “Eggs” is often used to refer to one-cell stage “embryos”. In the literature, injected “eggs” and one-cell stage “eggs” are typically “embryos”. Lines 87, 159, 161. The authors should clarify if CRISPR/Cas9 “constructs” are injected into embryos or CRISPR/Cas9 “components” such as RNA (sgRNA) and protein (Cas9). Lines 147 – 157 Parts of text can be revised to improve the flow of the article. For example, this section contains text that can be incorporated into the results section. Clarification regarding the generation of “knockouts” is needed. To make the claim that efficient mutagenesis is achieved to analyze the F0 for functional analysis, it must be clear how reliable the knockdown is. In the methods section, the authors write that “all positive mutants were heterozygous” (lines 203-204). The authors go on to state that “only one tyr mutant retained a wild-type allele” (line 281) in the results section. Were positive mutants heterozygous or did the authors find that the embryos lacked wild-type sequences when assaying their sequence clones from whole embryos? What is the authors definition of “heterozygous”? To get a real sense of how mosaic the F0s are, more than a dozen to one hundred sequences per fish should be analyzed. More clarification is needed in regard to how many clones were sequenced (See comments for lines 280 – 282 below). Line 242 – 247, 260 – 269. Parts of text can be revised to improve the flow of the article. Some text may be moved and incorporated in the discussion section. Line 280 – 282 The authors should include more information to justify their choice of methods and clearly demonstrate how reliable the knockdown is. I presume the authors selected the RH2B sgRNA target 1 clutch 3 embryos because they see high somatic activity (4/13, 30.8%). Is this the case? From the 4 embryos subjected to PCR screening for the RH2B gene, those PCR amplicons were subcloned and how many clones were sequenced? Figure 3A would suggest maybe 16 clones were sequenced. Is this also the case for tyr? According to the text, authors subcloned and sequenced tyr PCR amplicons from embryos that came from clutch 9 (line 279). However, Figure 3 described subcloned sequenced from clutch 8 (lines 316, 331). In regard to line 308, if the data positions the authors to do so, they can write that “no wild-type sequences were detected in any of the sequenced clones (n=# of sequenced clones).” However, lines 203-204 and 281 suggest the authors may be observing compound heterozygous fish. Line 292 – 294. A graphical presentation can be made for the indels generated. In a bar graph, a column may show how many in frame mutations and frameshift mutations were made that were deletions and another column of the insertions that were in frame or frameshift mutations. Another type of graph to depict the distribution of CRISPR/Cas9 induced mutations is to show number of mutations on the y axis and the base pair changes (- and +) plotted on the x axis. In this graph, authors will have bars that indicate deletion range and bars that indicate insertion sizes. Figure 2 In Figure 2C and Figure 2D, labels for which fragment is uncut and which are cleaved can be presented. The in vitro experiment is successful, but another control is to run the sgRNA alone. In Figure 2C, the large overexposed bands may be tyr1 and tyr2 sgRNAs and not cut DNA. References: 1. Salis P, Lorin T, Lewis V, Rey C, Marcionetti A, Escande ML, Roux N, Besseau L, Salamin N, Sémon M, Parichy D, Volff JN, Laudet V. Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish. Pigment Cell Melanoma Res. 2019 May;32(3):391-402. doi: 10.1111/pcmr.12766. Epub 2019 Jan 29. PMID: 30633441; PMCID: PMC6483885. Reviewer #3: The manuscript by Mitchell LJ et al. describes CRISPR-Cas 9 genome editing in anemonefish. The authors efficiently demonstrate successful editing of two separate target sites- RH2B and Tyrosinase producing gene at G0 of A.ocellaris embryos. The experiments are well designed, performed with appropriate controls and the manuscript is well written. The manuscript will be potentially important to study gene functions in other reef fishes. Comments: 1. Can authors comment on how is the CRISPR-Cas 9 protocol described here is novel and different from previously published CRISPR-Cas 9 editing done in other fishes? 2. Can this protocol be applied to precisely delete specific nucleotides at the target site and also Knock-in specific genes? 3. The authors show loss of function phenotype in tyr KO embryos, is there a functional phenotype to demonstrate loss of R2BH in the embryos? Probably by Immunohistochemical analysis of opsin trafficking in photoreceptors? 4. Comments on any off-target/unpredicted mutations. ********** 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 Reviewer #3: 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. |
Revision 1 |
CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants PONE-D-21-10870R1 Dear Dr. Mitchell, 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, Rajakumar Anbazhagan, Ph. D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 Reviewer #2: 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 Reviewer #2: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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) Reviewer #2: Reviewer Comments to Author(s): The revised manuscript describes the use of CRISPR/Cas9 for genome editing in reef fish or anemonefish. While CRISPR/Cas9 mediated mutagenesis has been performed in a previous publication (1), the rational is that no protocol has detailed how to inject anemonefish to generate mosaic mutants or complete knockouts in the F0 generation. In the revised manuscript (PONE-D-21-10870_R1), the authors established a system for implementing CRISPR/Cas9 to produce genome edited reef fish. Although the authors microinjection technique affected the survivability of injected embryos, the overall success rate of CRISPR/Cas9 mediated mutagenesis was measurable. Heritability of CRISPR/Cas9 induced mutations were not detected in the injected reef fish due to the model systems long generation time constraints. However, the authors generate mosaic founder (F0) embryos that closely resemble true null mutations, at least in the case of tyr. The ability to generate F0 mutants will allow testing the functional importance of genes, although off-target effect is more common with this approach. The research presented provides the groundwork for CRISPR/Cas9 application in anemonefish and has a greater impact on the advancement of the use of non-traditional model organisms in scientific discovery. Overall, the authors have addressed all comments, questions and concerns in the revised manuscript. The scientific arguments and interpretation are accurate and consistent with the results presented in the revised manuscript. The revised manuscript readability and flow of the text is presented with sufficient information for readers to follow the rationale, procedures and glean insights into the application of the CRISPR/Cas9 system for use on reef fish and other model organisms with longer generation times. References: 1. Salis P, Lorin T, Lewis V, Rey C, Marcionetti A, Escande ML, Roux N, Besseau L, Salamin N, Sémon M, Parichy D, Volff JN, Laudet V. Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish. Pigment Cell Melanoma Res. 2019 May;32(3):391-402. doi: 10.1111/pcmr.12766. Epub 2019 Jan 29. PMID: 30633441; PMCID: PMC6483885. ********** 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: No Reviewer #2: No |
Formally Accepted |
PONE-D-21-10870R1 CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants Dear Dr. Mitchell: 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 plosone@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. Rajakumar Anbazhagan Academic Editor PLOS ONE |
Open letter on the publication of peer review reports
PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.
We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.
Learn more at ASAPbio .