Review 1: "Engineering Probiotic Escherichia Coli Nissle 1917 to Block Transfer of Multiple Antibiotic Resistance Genes by Exploiting a Type I CRISPR-Cas System"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: The concept of engineering probiotic bacteria with CRISPR-Cas cassettes to reduce horizontal gene transfer is intriguing and has potential applications in various fields. In this study, the authors hypothesized that introducing a Type I-E CRISPR-Cas system into probiotic bacteria E. coli Nissle 1917 would restrict the horizontal gene transfer of antibiotic resistance genes, hence paving the way for safe use of such probiotic. Multiple aspects of the hypothesis were tested using different well-designed and well-controlled experiments. The CRISPR-Cas cassettes were provided both in trans as a separate plasmid or integrated into the bacterial genome. Both in vitro and in vivo performances were assessed and confirmed. Additionally, the activities of Cas3 and Cas9 were compared to support the choice of Cas3 cassettes. Overall, this study was well designed, the experiments were well executed, and the claims were supported by the results. This work helps fill a gap in the current research field regarding probiotic bacteria engineering.

Comments regarding the robustness of the study design include (1) the authors performed the competitive experiment of EcNs expressing Type I and Type II CIRSPR-Cas system, however, the competitive index against wild type EcN is missing. What level of fitness cost would integration of CRISPR-Cas cassette have? (2) was the adaptive nature of CRISPR-Cas tested, ie the capability to acquire new guide RNAs and target new ARGs? (3) where is the genomic integration site of the CRISPR-Cas cassettes (4) does the CRISPR-Cas cassette integration impact the local or global gene expression profile?

One minor comment regarding the clarity is: the main text could be improved as multiple derivative strains were created and different ones were used in different experiments, which could make it confusing to follow through.

Additional minor comments regarding the general application of the study involves evaluating the change in safety, immunogenicity, probiotic properties, and any additional adverse side effects after introduction of CRISPR-Cas cassettes. All in all, while engineering probiotic bacteria with CRISPR-Cas cassettes to reduce horizontal gene transfer holds promise for various applications, it's crucial to approach it with careful consideration of safety, ethical, regulatory, and ecological factors.