Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2

Jun Yang; Nilakshi Barua; Md. Nannur Rahman; Norman Lo; Tsz Fung Tsang; Xiao Yang; Paul K. S. Chan; Li Zhang; Margaret Ip

doi:10.1371/journal.pone.0261778

Peer Review History

Original SubmissionOctober 4, 2021
8 Nov 2021 Decision Letter - Yu-Hsuan Tsai, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-21-31939Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2PLOS ONE Dear Dr. Ip, 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 Dec 23 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. 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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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The baseline of the conclusion which chimeric crRNA improves the specificity of Cas12a-based detection of COVID-19 N501Y variant is acceptable and can be useful for the scientific community once the data is published. The following comments need to be addressed in the revision: 1. How does the LOD of 5,000 raw fluorescent units be selected? It seems that this is chosen in this paper with the intention to differentiate the detection result with crRNA 20-nt and chimeric crRNA 24-nt as false positive and negative, which makes the result more interesting. The more rigorous way to report specificity improvement using the fold change in background signal (e.g. ~ 3-fold) . Also, since the maximum signal of detection in chimeric crRNA also reduces, the author should compare the dynamic range of detection using different crRNA. 2. Statistical analysis of significance is not performed in all bar graphs where such analysis is needed and the method of statistical analysis is missing in the method section. Reviewer #2: Overall comments & recommendations: The aim of the study is the first report that chimeric crRNA could be useful for enhancing detection of the specificity of CRISPR-Cas12a for SARS-CoV-2 mutation N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without reducing its sensitivity. To test specificity, the authors developed N501Y chimeric crRNA 24-nt sequence and compared it with crRNA 20-nt to test N501Y variant and wild type of the virus. In addition, to determine whether the sensitivity of N501Y chimeric crRNA 24-nt is significant for N501Y detection, the limit of detection (LoD) was evaluated and compared to N501Y crRNA 20-nt using the tenfold serial dilution of synthetic RNA containing gene fragments of SARS-CoV-2. The study provided valuable data on this chimeric crRNA. However, there are some comments on the study. 1.In the background section, the authors should explain how application of chimeric crRNA on N501Y detection is significant. And, the authors should further describe how to identify the variants of Alpha, Beta, Gamma, and Mu. 2. Please describe Statistical analysis in Method. 3.In Discussion, the effectiveness and mechanism of chimeric crRNA on N501Y is needed to discuss. 4.Lines149: Please describe the SARS-CoV-2 origins in the experiment ****** 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: Yes:** Zhuobin Liang 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. Attachments Attachment Submitted filename: Comment.docx https://doi.org/10.1371/journal.pone.0261778.r001
Revision 1
16 Nov 2021 Author Response 12th November, 2021 To Professor Emily Chenette Editor in Chief, PLOS ONE Dear Prof. Emily Chenette, Submission of revised manuscript entitled “Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2” We enclose hereby the revised manuscript entitled “Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2” for considering to publish in your renowned journal PLOS ONE. We are very grateful for your comments and of the reviewers’. Our point-by-point response to the reviewers’ comments follow and our detailed revisions in the manuscript are highlighted with Tracked changes 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 Response: This manuscript has been prepared according to PLOS ONE’s style requirements and file naming format. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: [This work was supported by internal grants from the Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong.] We note that you have provided funding information that is 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: [This work was supported by internal grants from the Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong.] Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: For your kind information, the funding-related text, “This work was supported by internal grants from the Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong.” has been removed from the revised manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Response: We do not wish to change our Data Availability statement. 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. Response: ‘Data not shown’ has been replaced by Fig 2 and we have added the data in the revised manuscript. Please refer to line 123 in the revised manuscript with track changes. 5. 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. Response: The reference list is complete and correct. There is no retracted paper. We added Ref 10 and Ref 11 to the list. Please check the revised manuscript. For your kind information: 10. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019;14: 2986–3012. doi:10.1038/s41596-019-0210-2 11. Chen JS, Ma E, Harrington LB, DaCosta M, Tian X, Palefsky JM, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360: 436–439. doi:10.1126/science.aar6245 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 baseline of the conclusion which chimeric crRNA improves the specificity of Cas12a-based detection of COVID-19 N501Y variant is acceptable and can be useful for the scientific community once the data is published. The following comments need to be addressed in the revision: 1. How does the LOD of 5,000 raw fluorescent units be selected? It seems that this is chosen in this paper with the intention to differentiate the detection result with crRNA 20-nt and chimeric crRNA 24-nt as false positive and negative, which makes the result more interesting. The more rigorous way to report specificity improvement using the fold change in background signal (e.g. ~ 3-fold) . Also, since the maximum signal of detection in chimeric crRNA also reduces, the author should compare the dynamic range of detection using different crRNA. Response: Many thanks for your comments. It is very important issue you raised here. We have explained in the manuscript how 5000 AU was chosen as the threshold. For your kind information, below 5000 AU from the spectrophotometry reading, these test tubes are not visible under UV light. The signal of the N501Y and wild type of chimeric crRNA 24-nt is lower than that of crRNA 20-nt, so the fold change cannot give the exact picture of specificity in this case. That is why we have provided the data with 5000 AU as the threshold. We did not mention any dynamic range because when the signal is below 5000 AU, it cannot be visualized whereas at any value above 5000 AU, it has been visualized. 2. Statistical analysis of significance is not performed in all bar graphs where such analysis is needed and the method of statistical analysis is missing in the method section. Response: Thank you very much for your suggestions. Statistical analysis results have been added to all bar graphs. We also added the method of statistical analysis in the method section. Please check line 198 of the revised manuscript with track changes. Reviewer #2: Overall comments & recommendations: The aim of the study is the first report that chimeric crRNA could be useful for enhancing detection of the specificity of CRISPR-Cas12a for SARS-CoV-2 mutation N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without reducing its sensitivity. To test specificity, the authors developed N501Y chimeric crRNA 24-nt sequence and compared it with crRNA 20-nt to test N501Y variant and wild type of the virus. In addition, to determine whether the sensitivity of N501Y chimeric crRNA 24-nt is significant for N501Y detection, the limit of detection (LoD) was evaluated and compared to N501Y crRNA 20-nt using the tenfold serial dilution of synthetic RNA containing gene fragments of SARS-CoV-2. The study provided valuable data on this chimeric crRNA. However, there are some comments on the study. 1.In the background section, the authors should explain how application of chimeric crRNA on N501Y detection is significant. And, the authors should further describe how to identify the variants of Alpha, Beta, Gamma, and Mu. Response: Many thanks for your comments. As we are the first to use chimeric crRNA on detection in order to find a method that can improve the specificity of CRISPR-Cas12a detection, thus, we have added how the application of chimeric crRNA is significant in the discussion section. For your kind information: the sensitivity of chimeric crRNA is comparable to regular crRNA, whereas the specificity is higher and can be stable for more than 2 h. It improves and provides the versatility of applying CRISPR-Cas12a in a larger throughput which will take more time to prepare reactions, even as a point-of-care testing (POCT). Please refer to line 147 of the revised manuscript with track changes How to identify the variants of Alpha, Beta, Gamma, and Mu has also been added in the discussion. It is worthy to mention that, N501Y mutation is shared by Alpha, Beta, Gamma, and Mu; identification of N501Y is the first step of differentiating variants of Alpha, Beta, Gamma, and Mu from wild type and other variants. To further differentiate Alpha, Beta, Gamma, and Mu, other mutation sites in the Spike protein like 69-70 deletion or 144 deletion, 242-244 deletion or K417N, R190S or K417T, T95I or R346K can be detected respectively. Please refer to line 158 of the revised manuscript with track changes 2. Please describe Statistical analysis in Method. Response: Thank you very much for your valuable comments. We added the method of statistical analysis in the method section. Please check line 198 of the revised manuscript with track changes 3.In Discussion, the effectiveness and mechanism of chimeric crRNA on N501Y is needed to discuss. Response: Many thanks for your comments. The effectiveness and mechanism of chimeric crRNA on N501Y have been added in the discussion of the revised manuscript. For your kind information: The sensitivity of chimeric crRNA is comparable to regular crRNA, whereas the specificity is higher and it is stable for more than 2 h. Replacing the last 8-nt of the crRNA with DNA can decrease the binding energy between crRNA and target DNA, leading to less off-target of CRISPR-Cas12a [9]. RNA-guided Cas12a unleashes indiscriminate single-stranded DNase activity when CRISPR-Cas12a recognizes its target [11]. As the detection signal is produced by cleavage of fluorophore-quenched ssDNA fluorescent reporter, improving on-target specificity by chimeric crRNA 24-nt can increase the specificity of CRISPR-Cas12a detection. Please refer to line 147 and line 150 of the revised manuscript with track changes. 4.Lines149: Please describe the SARS-CoV-2 origins in the experiment Response: We have mentioned the SARS-CoV-2 origins in the revised manuscript (line 165). The above mentioned SARS-CoV-2 gene fragments was collected form BGI (Beijing Genomics Institute, a Chinese genome sequencing company, headquartered in Shenzhen, Guangdong, China). Yours sincerely, Margaret Ip Professor and Chairman, Department of Microbiology, The Chinese University of Hong Kong Email: margaretip@cuhk.edu.hk Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0261778.r002
7 Dec 2021 Decision Letter - Yu-Hsuan Tsai, Editor PONE-D-21-31939R1Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2PLOS ONE Dear Dr. Ip, 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 Jan 21 2022 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: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled '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: https://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, Yu-Hsuan Tsai Academic Editor PLOS ONE Journal Requirements: 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 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: Minor suggestion: Figure 1C and 1D and the new Figure 2A and 2C look very similar, and my understanding is that they show results from the same experiments but at different time points after reaction incubation. To clarify the difference between Figure 1 and Figure 2 results, I recommend adding the time-point information directly on the figures. [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.0261778.r003
Revision 2
8 Dec 2021 Author Response Response: Many thanks for your suggestions. Time-point information has been added to Figure 1C and 1D and the new Figure 2A and 2C. Please check the revised Figure 1 and Figure 2. Time-point information has also been added to the legends of Figure 1 and S1 Figure. Please check lines 82 and 267 of the revised manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0261778.r004
10 Dec 2021 Decision Letter - Yu-Hsuan Tsai, Editor Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 PONE-D-21-31939R2 Dear Dr. Ip, 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, Yu-Hsuan Tsai Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0261778.r005
Formally Accepted
15 Dec 2021 Acceptance Letter - Yu-Hsuan Tsai, Editor PONE-D-21-31939R2 Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 Dear Dr. Ip: 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. Yu-Hsuan Tsai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261778.r006

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