Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system

Daniel Addo-Gyan; Haruka Matsushita; Enya Sora; Tsubasa Nishi; Masao Yuda; Naoaki Shinzawa; Shiroh Iwanaga

doi:10.1371/journal.pone.0260176

Peer Review History

Original SubmissionNovember 2, 2021
13 Dec 2021 Decision Letter - Gordon Langsley, Editor PONE-D-21-34882Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system.PLOS ONE Dear Dr. Shiroh Iwanaga, 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. First, I would like to apologize for the time taken to arrive at a decision (Minor Revision) that was due to any potential qualified reviewers declining our invitation to review your submission. One person did accept and luckily she is a real expert in P. berghei chromosome modifications and so, I took the editorial decision based on only this single review. 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Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Summary The authors here present a study where they have generated a CRISPR/Cas9-based tool which facilitates the site-specific cleavage of Plasmoidum berghei chromosomes. By introduction of additional telomere and centromere elements together with the homology directed repair template, they can thereby generate transgenic parasite lines with split chromosomes that segregate and are faithfully maintained during cell division. The study builds upon improvements to the P. berghei CRISPR/Cas9 system that the some of the authors published earlier. They here report on the on the modifications to the system that enables chromosome splitting, the vector generation and proof of principle experiments where they split P. berghei chromosome 1 in two different (blood-stage) functionally redundant loci. They further report on the normal viability and lack of fitness effect for (one of the?) split chromosome 1 lines. Impact and quality This is a well written manuscript with clearly presented methods and results, and where conclusions are well supported by data. Data is of good quality and is underpinned by appropriate controls. The merit of this study is that it presents a for malaria parasites completely novel system to split chromosomes and generate viable progeny with split chromosomes. This tool in turn, as argued by the authors, makes it possible to study the effect of intra-nuclear spatial location of chromosomes, which the authors argue “may cause a gene expression and epigenetic state change: since telomere ends are invariably anchored to inner nuclear membrane”. They further suggest this will be a particularly useful tool to study the effect of intra-nuclear spatial localization on expression of multigene surface proteins and genes important for sexual development since they localize to the subtelomeric regions of malaria parasite chromosomes. The impact of the study is limited by that the authors present no evidence for change in intra-nuclear spatial location of split chromosomes (e.g. FISH) or of gene expression (e.g. RNA seq) due to chromosome splitting. Could one not instead argue that no change in gene expression is likely to occur due to chromosome splitting, since no observable change in phenotype is shown for the chromosome 1 split line? The impact of this system on our understanding of malaria parasite biology there for remain hypothetical. This type of data would significantly elevate the impact of the study. Nevertheless, that elevation would likely qualify it for a higher impact journal and I have no reservations in recommending the manuscript for publication as it is, with only minor revisions outlined below. Minor revisions 41 considered recently -> recently considered 95 this plasmid can be screened -> this plasmid can be selected? 103 perhaps will aid reader if you explain centric / acentric chromosome? 277 “… we compared the asexual multiplication of split-Ch1-S parasite to that of pbcas9”. Clarify here if split-Ch1-S is PBANKA_0111600 or PBANKA_0105300 line. Perhaps it is stipulated in methods or elsewhere but would be good to make more obvious. This goes for all figures too, it is not always immediately clear to the reader which split chromosome line you refer to. Fig S4 Mislabeling, two Panel D instead of E ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
18 Jan 2022 Author Response Reviewer’s Comments Reviewer #1: Summary The authors here present a study where they have generated a CRISPR/Cas9-based tool which facilitates the site-specific cleavage of Plasmoidum berghei chromosomes. By introduction of additional telomere and centromere elements together with the homology directed repair template, they can thereby generate transgenic parasite lines with split chromosomes that segregate and are faithfully maintained during cell division. The study builds upon improvements to the P. berghei CRISPR/Cas9 system that the some of the authors published earlier. They here report on the on the modifications to the system that enables chromosome splitting, the vector generation and proof of principle experiments where they split P. berghei chromosome 1 in two different (blood-stage) functionally redundant loci. They further report on the normal viability and lack of fitness effect for (one of the?) split chromosome 1 lines. Impact and quality This is a well written manuscript with clearly presented methods and results, and where conclusions are well supported by data. Data is of good quality and is underpinned by appropriate controls. The merit of this study is that it presents a for malaria parasites completely novel system to split chromosomes and generate viable progeny with split chromosomes. This tool in turn, as argued by the authors, makes it possible to study the effect of intra-nuclear spatial location of chromosomes, which the authors argue “may cause a gene expression and epigenetic state change: since telomere ends are invariably anchored to inner nuclear membrane”. They further suggest this will be a particularly useful tool to study the effect of intra-nuclear spatial localization on expression of multigene surface proteins and genes important for sexual development since they localize to the subtelomeric regions of malaria parasite chromosomes.  The impact of the study is limited by that the authors present no evidence for change in intra-nuclear spatial location of split chromosomes (e.g. FISH) or of gene expression (e.g. RNA seq) due to chromosome splitting. Could one not instead argue that no change in gene expression is likely to occur due to chromosome splitting, since no observable change in phenotype is shown for the chromosome 1 split line? The impact of this system on our understanding of malaria parasite biology there for remain hypothetical. This type of data would significantly elevate the impact of the study. Nevertheless, that elevation would likely qualify it for a higher impact journal and I have no reservations in recommending the manuscript for publication as it is, with only minor revisions outlined below. Response: Thank you for reviewing our manuscript to make it better. The details of the modifications according to your comments are described below. Minor revisions 1. 41 considered recently -> recently considered Response Thank you for pointing-out this grammatical error. We have modified the phase from “considered recently” to “recently considered” on line 41. 2. 95 this plasmid can be screened -> this plasmid can be selected? Response We have changed the word “screened” to “selected” on line 98. 3. 103 perhaps will aid reader if you explain centric / acentric chromosome? Response Thank you for this suggestion. We have included an explanation in parenthesis for both acentric and centric chromosomes. This can be seen from lines 108 to 112 as follows: This linear DNA fragment was used for HDR with the centric chromosome (Chromosome 1 fragment with the original centromere after cleavage) after cleavage by the Cas9-sgRNA complex. Another DNA fragment, which was used for HDR with the acentric chromosome (Chromosome 1 fragment lacking a centromere after cleavage), was amplified and cloned upstream of both telomere and centromere of pArm_R plasmid (S2 Fig)” (P.5,l.106-P.6,l.110) 4. 277 “… we compared the asexual multiplication of split-Ch1-S parasite to that of pbcas9”. Clarify here if split-Ch1-S is PBANKA_0111600 or PBANKA_0105300 line. Perhaps it is stipulated in methods or elsewhere but would be good to make more obvious. This goes for all figures too, it is not always immediately clear to the reader which split chromosome line you refer to. Response Thank you for this observation. In order to make it easier to understand which mutant parasite is being referred-to, the labeling for the mutants have been modified. For example, split-Ch1-S has been changed to split-Ch1-1116. “1116” indicates that the mutant was produced by splitting the parent parasite at the PBANKA_0111600 locus. Similarly, split-Ch1-L has been changed to split-Ch1-1053. Also, “1053” indicates that the mutant was produced by splitting the parent parasite at the PBANKA_0105300 locus. This change has been effected throughout the text and figures. 5. Fig S4 Mislabeling, two Panel D instead of E Response Thank you for pointing-out this mistake. The mislabeled “D” panel has been re-labeled as “E” in the S4 Fig caption. Attachments Attachment Submitted filename: Response to Reviewers_211224__SI.docx https://doi.org/10.1371/journal.pone.0260176.r002
9 Feb 2022 Decision Letter - Gordon Langsley, Editor Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system. PONE-D-21-34882R1 Dear Dr. Shiroh Iwanaga, 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, Gordon Langsley Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0260176.r003
Formally Accepted
14 Feb 2022 Acceptance Letter - Gordon Langsley, Editor PONE-D-21-34882R1 Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system. Dear Dr. Iwanaga: 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. Gordon Langsley Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260176.r004

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