Identification and characterization of two CRISPR/Cas systems associated with the mosquito microbiome
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The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host–microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.
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The work presented is clear and the arguments well formed.
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Dear Authors, thank you for addressing the comments from the referees. Before I can accept this work, I kindly ask you in your methods to clearly indicate for each bioinformatic tool you used, that you have done so by using default parameters (or any other parameters you have used). You have addressed this in your response to referees but I'd like you to include this information in the methods for the sake of helping other researchers that may want to use your papers and methods for analysis in other organisms. Whilst it seems intuitive, a less expert reader may not find the lack of specification to be a clear sign that default parameters were used.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature.
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Comments to Author
This study describes the identification of two CRISPR-Cas systems in Serratia Sp. Ag1 and investigates the origin of the spacers in each array. The introductory literature analysis provides an overview of the relevant CRISPR subtypes allowing the non-expert reader to understand the context of the paper findings. Overall, the materials and methods section is concisely written, potentially contributing to the loss of some detail. Though the reasoning for the below points may be obvious to a CRISPR-focused scientist, they may not be to a general audience. The figures and tables are sensible representations of the underlying data, though figure 3 may contain small typos. Throughout the results section there are a few points to address, but overall, the methods are likely sound, assuming proper replicates …
Comments to Author
This study describes the identification of two CRISPR-Cas systems in Serratia Sp. Ag1 and investigates the origin of the spacers in each array. The introductory literature analysis provides an overview of the relevant CRISPR subtypes allowing the non-expert reader to understand the context of the paper findings. Overall, the materials and methods section is concisely written, potentially contributing to the loss of some detail. Though the reasoning for the below points may be obvious to a CRISPR-focused scientist, they may not be to a general audience. The figures and tables are sensible representations of the underlying data, though figure 3 may contain small typos. Throughout the results section there are a few points to address, but overall, the methods are likely sound, assuming proper replicates of the RT-PCR experiments were undertaken. In terms of discussion of results, it is my view that the findings of the paper could explored further. Whilst there is discussion of the concordance of the results of this study and that of others looking at the E. coli type I-E system, there is no discussion of how this work's Type I-F results fit into the field, for example. Further, the repressor H-NS is mentioned, but there is no effort to put this into context of the results presented here. Is there a H-NS homologue in Serratia, for example? The authors should double check their grammar and conventions (I.e., ensuring italicisation of gene and species names) throughout to improve readability. Specific points to address: Line 20: reword to ensure that it isn't implied, as it currently is, that Serratia Sp. Ag1 is a novel bacterium. It is the discovery of the CRISPR-Cas systems that is novel. Line 59: Introduce the concept of a leader sequence Line 81: typo. Correct from "has" to have. Line 89: should read "between the gut" to improve sentence structure Paragraph starting line 111: How many repetitions of the RT-PCR experiments were undertaken? Was densitometry of the gel undertaken? If not, why? This would allow quantitative comparison between log and stationary growth, and statistical analysis of the results between replicates. Line 120-121; why were different numbers of cycles used for the control/ cas genes? Perhaps this is a lack of experience on my part. Line 126: What settings were used when CRISPR-Finder was used? Line 128: What settings were used when CRISPR-Target was used? Line 129: Is there any precedent for the ~70-75% matching nucleotides to identify spacer sequences? Line 131; how similar were the "20 similar sequences from different species"? Line 132; What settings were used within MEGA7? I am not familiar with the software, but one would expect additional information on the settings used would be required for replication. Line 142; Cluster 2 and Cluster 4 are not introduced in the text. What is the significance of this assignation? Line 148; as mentioned above, the details of the matches to other bacterial species Cas3 homologues should be provided. Line 163; clarification of text. "…could identify, most targets were" would read better as "…could identify, with most identified targets…" Line 165; remove "we reduced" so the sentence reads "…27/32 for CRISPR2 arrays), the number of hits reduced significantly" to improve clarity. Line 174; provide references. Line 186; STEC not defined Line 187-189; Again, this could be my lack of specialist knowledge, but I don't see why the presence of a phage record in NCBI suggests that acquisition events occurred recently? Please clarify. Line 191-194: Several typos; "Discrete" to "A discrete". "spaces" to "spacers". Remove "the", it is unnecessary. Provide references for the Enterobacteriaceae data. Line 200-201: What value is this sentence adding as it is? What is the context in Serratia? Is there any context for the Type I-F system? Line 216: reword to "recurrent encounters between phages and symbiotic bacteria" Line 217: reword to "demonstrated that phage infection can alter bacterial levels" if appropriate. Line 219: Interact, not interaction. In Figure 3, the X axis reads CRISPR1, CRISPR2, and CRISPR3. What is CRISPR3? Is this a typo where the CRISPR 2 field should read CRISPR 2.1 and CRISPR 3 read CRISPR 2.2? If not, please explain the labelling more comprehensively in the figure legend. Line 434 typo; "closes BLAST-p" ought to read "closest" Line 439 in the Fig 3 figure legend, there is a typo. "CRISP loci" should read CRISPR loci In table 2 Some target species names are marked with an asterix. What is the significance of this? It seems to relate to fields where the target species is listed as phage, but the organism suggests the sequence is that of a plasmid? Please address this in the table legend or resolve. Also in table 2, the nucleotide ID is listed as a fraction. It would be more helpful if this were a percentage.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
This manuscript describes the crispr loci of a strain of Serratia associated with the mosquito gut. There are two type I crispr systems and 3 associated CRISPR loci. The genes are shown to be transcribed more highly during exponential than log phase. Perhaps the most interesting aspect of this paper is the observation that crispr 1 seems to be mostly targeting plasmids while 2.1 and 2.2 mostly target phage. Given the small number of spacers and the numbers that don't match well to any target, this is a bit hard to interpret the significance of this. Overall, this is a short, descriptive paper that represents a rather incremental contribution to the literature. One suggestion to strengthen it without too much extra work would be to analyse the crisprs of the highly related Ag1 system to determine …
Comments to Author
This manuscript describes the crispr loci of a strain of Serratia associated with the mosquito gut. There are two type I crispr systems and 3 associated CRISPR loci. The genes are shown to be transcribed more highly during exponential than log phase. Perhaps the most interesting aspect of this paper is the observation that crispr 1 seems to be mostly targeting plasmids while 2.1 and 2.2 mostly target phage. Given the small number of spacers and the numbers that don't match well to any target, this is a bit hard to interpret the significance of this. Overall, this is a short, descriptive paper that represents a rather incremental contribution to the literature. One suggestion to strengthen it without too much extra work would be to analyse the crisprs of the highly related Ag1 system to determine whether they are related to the Ag2 crisprs, and whether there is a similar bias in spacer origin. Specific points: 1. In figure 1, indicate that there is a fused Cas2-Cas3 gene for type I-F crispr 2. P3 line 53. The term "small" here is not clear - small in relation to what? They are large in comparison with most bacterial immune systems. 3. P7 line 166 - please check this sentence as it looks like there's a problem. 4. The RT-PCR analyses seem to have been carried out only once. It would be preferable to repeat this to provide more confidence in the data and conclusions drawn. Malcolm White
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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