Dear Dr Yin,

Thank you for submitting your manuscript entitled "The compact high-fidelity EbCas12a enables all-in-one AAV delivery system" for consideration as a Research Article by PLOS Biology. Please accept my apologies for the delay in getting back last week as we consulted with an academic editor about your submission.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review.

IMPORTANT: After discussions within the editorial team, we would like to consider your manuscript as a Short Report. Upon resubmission (details below), I would be grateful if you could tick 'Short Report' as the article type.

Before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Aug 15 2023 11:59PM.

If your manuscript has been previously peer-reviewed at another journal, PLOS Biology is willing to work with those reviews in order to avoid re-starting the process. Submission of the previous reviews is entirely optional and our ability to use them effectively will depend on the willingness of the previous journal to confirm the content of the reports and share the reviewer identities. Please note that we reserve the right to invite additional reviewers if we consider that additional/independent reviewers are needed, although we aim to avoid this as far as possible. In our experience, working with previous reviews does save time.

If you would like us to consider previous reviewer reports, please edit your cover letter to let us know and include the name of the journal where the work was previously considered and the manuscript ID it was given. In addition, please upload a response to the reviews as a 'Prior Peer Review' file type, which should include the reports in full and a point-by-point reply detailing how you have or plan to address the reviewers' concerns.

During the process of completing your manuscript submission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

ORCiD I plosbio.org I @PLOSBiology I Blog

California (U.S.) corporation #C2354500, based in San Francisco

Dear Dr Yin,

Thank you for your patience while your manuscript "The compact high-fidelity EbCas12a enables all-in-one AAV delivery system" was peer-reviewed at PLOS Biology. Please accept my sincere apologies for the delays that you experienced during the peer review process. Your manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports.

As you will see, the reviewers are generally positive about the findings but Reviewer #1 notes that the overall strength of the biochemical characterization of enEbCas12a should be strengthened. Specifically, this includes analysing Cas12a variants to compare editing efficiency and using deep sequencing to more robustly characterize potential off-target effects. After discussions with the Academic Editor, we will not make addressing major comment #2 essential for the revision but we encourage you to include this data if possible.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

We expect to receive your revised manuscript within 3 months. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may withdraw it.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point-by-point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Revised Article with Changes Highlighted" file type.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

------------------------------------

REVIEWS:

Reviewer #1: Wang et al. demonstrated genome editing using a compact Cas12a, EbCas12a. They determined the TTTV PAM sequences of EbCas12a and developed engineered version of EbCas12a, enEbCas12a. Next, the activity and specificity of enEbCas12a were compared with compared with AsCas12a and LbCas12a. Lastly, the authors investigated a single AAV system with EbCas12a and crRNA in vitro and in vivo.

1. In Figure 3E, the activity of enEbCas12a was compared only with wildtype AsCas12a and LbCas12a at seven target sites. To establish the utility of enEbCas12a, it would be necessary to measure and compare editing efficiency at a sufficient number of target sites where statistical significance can be demonstrated. I strongly recommend including the latest Cas12a variants, such as enAsCas12a (PMID = 30742127) or Lb2Cas12a (PMID = 33738137), for comparison.

2. If possible, it would be beneficial to perform an analysis of the features of target sites where enEbCas12a exhibits higher efficiency compared to other Cas12a variants.

3. To comprehensively analyze off-target effects, it's crucial to ensure comparable on-target activities. In Figure 3F, we require a clear description of the on-target activities for AsCas12, LbCas12a, and enEbCas12a. It would be more accurate to measure indel frequencies at endogenous target sites rather than relying solely on Guide-seq read counts. Furthermore, to validate the identified off-target sites using Guide-seq, it's essential to confirm the presence of indels at these potential off-target sites through deep sequencing.

4. Although enEbCas12a is relatively small with a size of 1158 amino acids compared to the Cas12a family, there have been much smaller genome editing tools developed recently, such as Un1Cas12f (529 aa, PMID = 34475560) and AsCas12f (422 aa, PMID = 37400536). Among Cas9 variants, sRGN3.1 (1057 aa) and SlugCas9 (1054 aa) that showing robust activity (PMID = 37188955) also smaller than enEbCas12a . Therefore, an analysis and discussion of the advantages that enEbCas12a possesses compared to these smaller counterparts is needed.

Reviewer #2: The authors have cloned and harnessed an enhanced variant of EbCas12a and demonstrate its compatibility with AAV vectors in cultured cells and in the mouse liver. The data are encouraging as their Cas12a version alleviates previous concerns about the size of Cas12a that have hampered packaging in and delivery by AAV.

Overall, this is an interesting article and a useful advance, thus I only have a few minor comments:

1) Did the EbCas12a vector package well? What were the average titers as compared to other vectors in the authors' hands?

2) Which dose was actually delivered to the mice? It says 2e11 in the text but 5e11 in the figure, and I couldn't find additional information in the Methods.

3) The drop in cholesterol levels in the mice is not overly impressive albeit significant. It would be good if the authors could further support and illustrate vector efficacy by showing the results of T7E1 assays from the mouse livers and quantifying the extent of target editing. Does it correlate with the mild drop in cholesterol?

Dear Dr Yin,

Thank you for your patience while we considered your revised manuscript "The compact high-fidelity EbCas12a enables all-in-one AAV delivery system" for publication as a Short Report at PLOS Biology. Please accept my apologies for the delays that you have experienced during this round of the peer review process. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

Based on the reviews, I am pleased to say that we are likely to accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests that I have provided below (A-G). In addition, we ask that you please improve the quality of the writing/language in the Abstract before publication, by enlisting the services of a professional editing services or the help of an English-speaking colleague:

(A) We would like to suggest the following modification to the title:

““Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system”

(B) In the animal ethics statement provided in the Methods section, please provide the specific approval number issued by the Institutional Animal Care and Use Committee of Wuhan University.

(C) You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

-Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

-Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Figure 1E-F, 2A-B, 2F, 3B-F, 4D, S7B, S8B, S9B, S13B-C, S14B-C

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

(D) Thank you for depositing the GUIDE-seq in the GEO database (GSE236647), but I would be grateful if the deep sequencing data could also be included in this deposition.

(E) Please also ensure that each of the relevant figure legends in your manuscript include information on *WHERE THE UNDERLYING DATA CAN BE FOUND*, and ensure your supplemental data file/s has a legend.

(F) We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in the following Figures:

Figure 1C, 2C, 2E, 4B-C, S1B, S3, S7A, S8A, S9A, S13A, S14D

We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

(G) Please ensure that your Data Statement in the submission system accurately describes where your data can be found and is in final format, as it will be published as written there.

------------------------------------------------------------------------

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Please do not hesitate to contact me should you have any questions.

Kind regards,

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

------------------------------------------------------------------------

Reviewer remarks:

Reviewer #1: In the revised manuscript, the authors have conducted a comprehensive evaluation of the compact, high-fidelity EbCas12a nuclease through additional wet experiments and analysis. The AAV-enEbCas12a described in the study would improve the efficacy and specificity of gene therapy. Thus, I recommend considering this study for publication in PLOS Biology.

Reviewer #2: I thank the authors very much for fully addressing my comments.

Dear Lei,

Thank you for your patience while we considered your revised manuscript "Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system" for publication as a Short Report at PLOS Biology.

Following on from our previous correspondence, I am writing to ask that you please provide some additional reporting details about the EbCas12a sequence in the manuscript before we can move forward to publication. I have listed these requests below (A-C), but please do let me know if you have any further questions about this:

(A) Thank you for clarifying that the EbCas12a sequence has been obtained from a metagenome-assembled genome of a currently unclassified Erysipelotrichia isolate. In the Genbank accession, it appears that this sequence was originally obtained in an metagenomic study in which the authors sequenced wastewater from ponds of two small diary industries in Argentina (BioProject: PRJNA508305). This dataset was initially deposited in 2020 and then eventually published in the PeerJ in 2022 (Irazoqui et al, 2022, PeerJ, PMID 35310160). At this time, we ask that you please and cite and refer to/contextualize this prior work in the PeerJ that initially provided the metagenomic sequences in the manuscript.

(B) Pleaser ensure you refer to the EbCas12a as being obtained from the metagenome-assembled genome of a currently unclassified Erysipelotrichia in the manuscript text. This will need to be changed throughout the manuscript text, figure legends, abstract etc where the text currently refers to "the Erysipelotrichia bacterium bacteria" or "the Erysipelotrichia bacterium". Specifically, we would suggest that you state that the Cas12a was obtained from the metagenome-assembled genome of a currently unclassified Erysipelotrichia (eg the first time in the paper and then in the Methods section), and then when it is subsequently referred to you can write "a Cas12a ortholog from a bacterial species of the class Erysipelotrichia" or similar.

(C) Please include the Genbank and Taxonomy ID accession numbers in the Data Availability Statement in the online submission form.

------------------------------------------------------------------------

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Kind regards,

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

ORCiD I plosbio.org I @PLOSBiology I Blog

California (U.S.) corporation #C2354500, based in San Francisco

Dear Lei,

Thank you for the submission of your revised Short Report "Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Bon-Kyoung Koo, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Best wishes, 

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

ORCiD I plosbio.org I @PLOSBiology I Blog

California (U.S.) corporation #C2354500, based in San Francisco