Construction of an arrayed CRISPRi library as a resource for essential gene function studies in Streptococcus mutans

ABSTRACT Arrayed mutant libraries are important resources that have advanced our understanding of bacterial genetics. Until recently, essential genes which are necessary for bacterial survival, and constitute ~10% of bacterial genes, were not included in arrayed library resources. However, recent advances in clustered regularly interspaced short palindromic repeats interference (CRISPRi) have made it possible to study essential genes with increasing throughput. Here, we constructed an arrayed CRISPRi library in the dental caries pathogen Streptococcus mutans, known as SNAP ( S treptococcus mutans arrayed CRISPRi). In this library, each strain contains a short guide RNA which selectively targets a single essential gene, and this is controlled through xylose induction. In total, the library can selectively repress >250 essential and growth-supporting genes. Initial characterization showed that SNAP strains grow as expected, and initial proof-of-concept experiments displayed the utility of the library. We anticipate that this library will be of benefit to the research community by allowing for high-throughput characterization of S. mutans essential genes. IMPORTANCE The construction of arrayed mutant libraries has advanced the field of bacterial genetics by allowing researchers to more efficiently study the exact function and importance of encoded genes. In this study, we constructed an arrayed clustered regularly interspaced short palindromic repeats interference (CRISPRi) library, known as S treptococcus mutans arrayed CRISPRi (SNAP), as a resource to study >250 essential and growth-supporting genes in Streptococcus mutans. SNAP will be made available to the research community, and we anticipate that its distribution will lead to high-quality, high-throughput, and reproducible studies of essential genes.

1. Essential genes are genes that an organism requires to survive under specific conditions.In this study, the essential gene functions can be induced-terminated.The identified genes are growth-associated or growth-supporting but not essential since the genes are not completely knocked out, and the growth is not entirely forbidden.As the author also mentioned, the leaking function of CRISPRi may work to make the organism survive.Gene knock-out should be performed with several positive controls to confirm the results.2. How can you evaluate the target-off effect of CRISPRi?Do the results are validated through gene expression or protein expression levels?3. Line 39-40, one more reference could be added here (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216606/).4. Line 84, the function of perR gene in Kajfasz's study should be described in the introduction.5. Line 106, the method to calculate the AUCsgRNA/AUCcontrol values and how to get the p-values should be stated.Multiple testing corrections of the p-values should be performed.6. Line 110, please add an SI table to show the AUCsgRNA/AUCcontrol values of the 141 strains.8. Line 138, please explain the rationale for the threshold setting as 15%.An outlier analysis could be performed in the w/o xylose data to figure out a better threshold.The same comment is for the '0.5 increase' at line 142.9. What does each dot mean in Fig. 1 and 2B?Is that the mean or median value of a strain?How many replicates are there for each mutant?Please state the information in the legends.
Reviewer #2 (Comments for the Author): In this study, the authors describe the construction of an arrayed CRISPRi library of Streptococcus mutans UA159 and do some fundamental characterization and proof-of-concept experiments.This library will be a useful tool for the field.Overall, the manuscript is well-written, logical, and the conclusions are supported be the results.The content of the article also fits nicely with the scope of mSpectrum.Improvements needed prior to publication are mainly minor in nature, but will significantly improve clarity and readability for the audience.These improvements are listed point by point below: Line 65: there should be a comma after "expected" Figure S1: The black text is nearly impossible to read in some of the dark backgrounds.Please make the background color lighter or more transparent and/or change the text to white in these cases to improve readability.Figure S3: Again, please add titles to the graphs.In the legend, I believe the panel letter should come before the text describing the panel, not after (titles will help clear that up, in any case).The graph legends are also confusing...the concentrations are for the antimicrobial added?Or inducer?Please make all this more clear.
Figure S4A: The order of concentrations in the legend makes no sense, please rearrange so they are in order of increasing.Also please add titles.Also, these are in mM, while the main text discusses things in terms of µg/ml, please make them all consistent.Also, if by 125 mM you mean 125 µg/ml, this does appear to inhibit growth, and therefore is not a sub-MIC concentration, as described in the main text? Figure S4B: So the concentration used in the main experiment was 0.25 µg/ml, but that is not one of the growth curves shown?Since there did appear to be a minor lag of growth with 0.15625 and certainly a lag with 0.3125, please explain how you justify calling 0.25 a sub-MIC concentration.
Line 186: Why not test with higher NaF concentration where you might expect to see more impact and determine which genes are affected/impacted?Figure 3: Again, please increase the size of the text relative to the figure.Is 1742 fabK?fabK is called out in the main text on line 195, but doesn't appear to be labeled as such in this graph.
General comment: Please speculate on why you do not see reduced growth with the basal repression.You spend Figure 1A showing that there isn't reduced growth without xyl, but then suggest transcription of many genes is likely repressed enough to increase sensitivity to antimicrobials.I think this is worth a sentence or two to reconcile.

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In this study, the authors constructed an arrayed CRISPRi library to study the essential genes related function in growth, biofilm formation, and antibiotic resistance in S. mutans.The innovative method is promising in the discovery of potential drug targets and study essential gene functions that usually cannot obtained.This work could be very useful.However, there are several weaknesses should be addressed before allowing for publication.
Major comments: 1. Essential genes are genes that an organism requires to survive under specific conditions.In this study, the essential gene functions can be induced-terminated.
The identified genes are growth-associated or growth-supporting but not essential since the genes are not completely knocked out, and the growth is not entirely forbidden.As the author also mentioned, the leaking function of CRISPRi may work to make the organism survive.We thank the reviewers for their comments on the manuscript and have edited the manuscript to address their concerns.
A point-by-point response follows: Response to Reviewer #1: Comment 1: Essential genes are genes that an organism requires to survive under specific conditions.In this study, the essential gene functions can be induced-terminated.The identified genes are growth-associated or growth-supporting but not essential since the genes are not completely knocked out, and the growth is not entirely forbidden.As the author also mentioned, the leaking function of CRISPRi may work to make the organism survive.Gene knock-out should be performed with several positive controls to confirm the results.Response.Our reason for targeting essential/growth supporting genes with CRISPRi stems from prior research using Tn-seq.We realized that this was not introduced, and we have added a brief description starting at Line 78: "Our basis for targeting these genes with a CRISPRi system was conceived from prior transposon sequencing (Tn-seq) efforts exploring gene essentiality in S. mutans (17)."Note, that during our work with Tn-seq we validated >10 growth supporting genes using gene mutagenesis.
7. Line 133, are the comparisons shown in Fig 2A between w/o xylose and 0.5% (or 1%) xylose of the sgRNAgtfB strain?What are the p-values of the comparison between w/o xylose and 0.5% (or 1%) xylose in the sgRNAscrB and w/o sgRNA strains?

Figure 1 :
Figure 1: I would suggest adding a header/title above each volcano plot so the reader can easily and quickly figure out what they are looking at.The size of the text relative to the figure should also be increased for readability.

Figure 2 :
Figure2: Again, the size of the text is way too small relative to the figure to be readable...I printed out the document and I can't read it at all...I have to zoom way in on the computer.Also, the text in all figures is pixelated and low quality when you do zoom in.

Figure 2 :
Figure 2: The only comparisons that are statistically significant the two shown with ***?? None of the others are (w/o sgRNA w/o xylose vs 0.5 xylose, for example)?? Please clarify this and make sure it is clear to the reader which comparisons in the figure are and are not statistically significant.Lines 141-143: This point is a bit confusing.Please explain what you mean here a little better, this is with or without xylose and what do you mean "by expected"?
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Re: Response to Reviewer's Comments for Spectrum03149-23
Gene knock-out should be performed with several positive controls to confirm the results.2.How can you evaluate the target-off effect of CRISPRi?Do the results are validated through gene expression or protein expression levels?//www.ncbi.nlm.nih.gov/pmc/articles/PMC3216606/).4.Line 84, the function of perR gene in Kajfasz's study should be described in the introduction.5.Line 106, the method to calculate the AUC sgRNA /AUC control values and how to get the p-values should be stated.Multiple testing corrections of the p-values should be performed.6.Line 110, please add an SI table to show the AUC sgRNA /AUC control values of the Line 138, please explain the rationale for the threshold setting as 15%.An outlier analysis could be performed in the w/o xylose data to figure out a better threshold.The same comment is for the '0.5 increase' at line 142.9.What does each dot mean in Fig.1 and 2B?Is that the mean or median value of a strain?How many replicates are there for each mutant?Please state the information in the legends.