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  4. Reviewer #3 (Public Review):

    The authors clearly demonstrate the effectiveness of optimized tools to generate precise C to T point mutations in zebrafish F0 embryos. The demonstrate germline transmission and an associated mutation for one mutation. There is sufficient data for members of the community to consider adopting these tools to generate mutation in their own laboratories

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  5. Reviewer #2 (Public Review):

    Rosello et al. present very compelling evidence that Cytosine base editors can be used to introduce G:C to A:T base conversions with high efficiency in zebrafish. Furthermore, they describe engineering and validation of a base editor targeting the NAA PAM sequence. Finally, they have developed a potential novel model of the Noonan syndrome. The manuscript represents an important and much needed advance in precision genome editing in the zebrafish model system.

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  6. Reviewer #1 (Public Review):

    The manuscript by Rosello et al., describes the application of cytosine base editing to efficiently introduce known and predictable mutations into disease genes in vivo in zebrafish, and examine signaling pathways and model disease. The majority of the data presented is analysis of editing precision and efficiency in somatically targeted embryos, with one example of a precise edited germline allele recovered. A direct comparison of the cytosine base editor BE4 and an improved version ancBE4max indicates both are highly efficient at somatic base editing. ancBE4max reduces alteration of bases outside the base editing window, and the data suggests loci for which BE4 base editing has failed can be targeted with ancBe4max. The authors demonstrate efficient base editing in embryos at multiple cancer genes (up to 91%), introducing activating mutations into oncogenes and nonsense mutations in a number of tumor suppressors. A S33L allele was introduced into the b-catenin gene ctnnb1 to activate the wnt signaling pathway as evidenced by expression of the wnt reporter Tg(tcf:GFP). Another novel aspect of this study is that the authors have expanded base editing target site selection by switching out the ancBe4max SpCas9 PAM-interacting motif domain with the domain from Spymac, which recognizes an NAA PAM. ancBe4maxSpymac editing efficiency was modest (16-19%). The method reported here has strong potential for effective combinatorial mutagenesis to map complex genetic interactions that underly disease pathogenesis. Overall, this study demonstrates cytosine base editing is an efficient and powerful method for introducing precise in vivo edits into the zebrafish genome.

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  7. Evaluation Summary:

    The manuscript by Rosello et al. represents a major advance in implementation of cutting-edge genome editing methodologies in the zebrafish. The study seeks to describe optimized tools for precise base editing in zebrafish and to demonstrate their effective application. Overall, this study demonstrates that cytosine base editing is an efficient and powerful method for introducing precise in vivo edits into the zebrafish genome, and will be of interest to scientists who use zebrafish and other genetic systems to model human development and disease.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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