Dear Dr Grigoraki,

Thank you very much for submitting your Research Article entitled 'CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and interaction with metabolic enzymes.' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some concerns about the data presented in Figures 2&3. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a revised version. We cannot, of course, promise publication at that time.

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[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Subba Reddy Palli, Ph.D.

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Acceptable for publication. Would have been nice to see LD50 data for the insecticides.

Reviewer #2: This manuscript describes the use of genome editing to introduce the pyrethroid resistance mutation L1014F into the VGSC of Anopheles gambiae. The impact of this on insecticide sensitivity and organismal fitness was assessed. Furthermore the interaction of this mutation with a metabolic mechanism, over-expression of glutathione transferase Gste2, was investigated.

The manuscript is very well written and the study innovative and important. The kdr mutation has been linked to resistance in An gambiae for more than two decades, however, the precise level of resistance conferred by this mechanism, in isolation or combination with other mechanisms, has remained unclear. This study emphatically demonstrates its importance in conferring resistance while also uncovering fitness costs associated with the mutations presence.

The results are thoroughly and carefully discussed and any potential limitations/confounding effects (i.e. from off-target effects of CRISPR-CAS) noted.

In summary I recommend this paper is accepted and the following typos corrected in production.

Line 192: change: Mutation L1014F carries fitness costs. To ‘The L1014F mutation carries fitness costs’.

Line 261: correct the grammer in the following sentence: The resistance observed for Kisumu-F/F against permethrin and deltamethrin are very similar to the levels of insensitivity mutation L1014F provides to Ae. aegypti VGSCs expressed in the in vitro Xenopus oocyte system (8-fold for permethrin and 14-fold for deltamethrin) (29).

Reviewer #3: In this paper, Grigoraki et al. generate an Anopheles gambiae transgenic line bearing a kdr mutation to further validate that kdr contributes to insecticide resistance. Using CRISPR/cas9 system, they edit the genome of a susceptible mosquito strain to introduce single nucleotide polymorphism (kdr mutation L1014F). They further test the mortality of this line using different insecticide compounds and confirm kdr mutation L1014F increases resistance to insecticides. Overexpression of gste2, a detoxification enzyme, in the kdr mutant line increase the resistance phenotype. Finally, they analyse the effect of the mutation on mosquito fitness. This paper is well written, clear and conclusions supported by the data. However, I do not think that this study reveals novel methods nor biological insights as it only confirms previous studies showing kdr is involved in resistance to insecticides and therefore, may be better suited for a more specific journal.

Minor comments

Why is there a lot of variation in F/F mosquitoes mortality after ITN exposure (Fig 3) with some mosquito groups showing a mortality above 90%, ie are susceptible?

The resistance to deltamethrin varies a lot as well (eg fig2 with 70-80% mortality vs fig4 with less than 20%). How can this be?

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Dear Dr Grigoraki,

Thank you very much for submitting your Research Article entitled 'CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes.' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and the editors ask that you address the comment from Reviewer 1 of the previous version and include the LD50 data for insecticides.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that 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 to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Subba Reddy Palli, Ph.D.

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Dear Dr Grigoraki,

We are pleased to inform you that your manuscript entitled "CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes." has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Subba Reddy Palli, Ph.D.

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

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Comments from the reviewers (if applicable):

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