A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps. IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.

Dear authors, the manuscript entitled "A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis" is a manuscript with general interest for the scientific community working with B. subtilis. In general, it is well written but can be rephrased in some parts to make it straighter. I think the title is a bit misleading, since the approach is not suitable for the wild type strain, thus it is only applicable for the BKE collection, what should be mentioned in the title. Targeting the erm-gene is quite obvious for this collection. The manuscript describes different modifications which were performed, and it is sometimes hard to follow them, and to see what the outcome is. I would suggest to rewrite the description of the different experiments to make it clearer what was done and what is really the novel approach. In general, the manuscript is written well but very often sought is used, what might be replaced. Also the efficiency by using gDNA or PCR fragments is quite low compared to the cloned HR repair templates, this should be discussed a bit more.
Minor: -In table 1 there is missing a space in-between left + right -What is the reason for the different length of the US and DS fragments? -Lane 220 is it really high yield, or is it more efficiency? -Lane 235 how can a construct itself be successful? Would rewrite that in ....successfully deleted or so on... -Lane 417, as described in 817), would use the author name here.
Reviewer #2 (Comments for the Author): Summary Sachla et al. describe a simplified method to modify the Bacillus subtilis genome using CRISPR-Cas9 editing and a pre-existing collection of B. subtilis strains with individual genes replaced by erythromycin (erm) resistance cassette (BKE collection). They design a guide RNA that targets the erm cassette and allows Cas9-mediated cleavage at the erm gene in any of the BKE knockout strains, and through homologous recombination a DNA repair template modifies the genome at the cleavage site. They demonstrate the application of this method to several genome-editing examples. While CRISPR-Cas9 editing was already established in B. subtilis, this method removes the guide RNA design step by only requiring the erm guide established in this paper. The editing plasmid and methods presented here are a valuable resource for the B. subtilis field that will speed strain construction and improve flexibility in introducing mutations.
General comments 1. The % efficiency for MLSS is 100% for all "cloned repair template" results but is very low for "cotransformation with repair template" (Table 1). This high failure rate by co-transformation is surprising, but the authors do not address the cause of this issue. Why is there a large fraction of MLSS clones that do not have the expected genotype? Is it possible that larger deletions flanking the erm gene occurred, or is there another explanation? I think other researchers may be reluctant to attempt the co-transformation approach if they aren't sure of the outcomes-the authors should try to address this by genotyping a few of the unexpected MLSS mutants. 2. The paper assumes that the audience has extensive knowledge of B. subtilis and has a solid understanding of CRISPR-Cas9 experiments. I appreciate the workflow detail in Supp Fig S1; however the authors should also write out each step in the S1 figure legend to further clarify what is happening at each step, particularly for readers with less background knowledge on B. sub and CRISPR. Additionally, when they mention Fig 1 in the text, they should also reference that a more detailed explanation of the process can be found in S1.
Specific comments 1. In lines 180-186, the way the text is written suggests that 42{degree sign} C growth causes the loss of erm resistance; however this actually causes the loss of plasmid (kan resistance). It would be helpful to break these steps apart to make it clear that the loss of erm resistance comes from CRISPR repair at 30{degree sign} C, with a separate sentence explaining loss of plasmid (kan) comes from growth at 42{degree sign} C. Additionally, the efficiency of loss of the plasmid is never mentioned but is important information to include. 2. In line 287 the authors briefly mention genetic congression, but their explanation was incomplete. The authors should mention how congression could affect the outcomes of the experiment, such as with using gDNA as a repair template. 3. In line 143, the title "Genome-editing using pAJS23 with a cloned guide RNA" should probably read "a cloned repair template" instead. 4. There may be a typo in the second to last row of Table 1, the calculation for % efficiency for MLSS used 3/28 but the previous column indicated that 26 colonies were tested.

Reviewer #3 (Comments for the Author):
A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis Authors (Sachla AJ et al.) would like to report a simple method for genetic manipulation of Bacillus subtilis. Authors designed a CRISPR/Cas9 system that recognizes the erythromycin cassette gene commonly found in the individual gene KO collection of Bacillus subtilis. Authors carried out gene replacement between erythromycin resistance cassette and various designed DNAs (plasmids, PCR products etc.) for FLAG-tag fusion, single point mutation, non-coding region changes, GFP fusion, markerless deletion. This study will be helpful in the systematic analysis of genetics, physiology, and metabolism of Bacillus subtilis. However, authors need to address the recent advances of CRISPR/Cas technologies in the engineering of microbial genomes, and to describe pros and cons of authors' new system, compared to other studies of CRISPR/Cas microbial genome engineering.
Following points need to be addressed.
Line 41, "with no requirement for intermediate cloning steps" can be deleted because "this strategy bypasses the need for gRNA design" is already stated in the same sentence.
Line 52, "free of off-target effects" can be deleted because genomic complexity is not high in the microbial genome. Authors don't need to emphasize "free of off-target effects" in the microbial system.
Line 265 Single nucleotide genome editing can be achieved by oligo-directed mutagenesis followed by negative selection of CRISPR/Cas9 or Cpf1. Authors need to refer to one of the references (PMID 34261850, 34208669, 32807756, 32327447).

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Reviewer #1 (Comments for the Author):
Dear authors, the manuscript entitled "A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis" is a manuscript with general interest for the scientific community working with B. subtilis. In general, it is well written but can be rephrased in some parts to make it straighter. ** Thank you for your supportive comments. We have edited the text for clarity as noted below and as shown in the "marked" version that is uploaded.
I think the title is a bit misleading, since the approach is not suitable for the wild type strain, thus it is only applicable for the BKE collection, what should be mentioned in the title. Targeting the erm-gene is quite obvious for this collection. ** We considered modifying the title, but decided against it. Very few scientists, even in the B. subtilis community, work directly in the "BKE" collection of strains, usually because each lab has its own wild-type (parent) strain. As a first step, it is always best to move erm mutations in your favorite gene (i.e. yfg::erm) into your own lab's wild-type strain. This might be a version of the type strain B. subtilis 168, but could also be any of a number of related strains. Historically, scientists from the Losick lab use "PY79", from the Hoch lab "JH642" and those studying biofilms and motility may use the "wild" isolate B. subtilis 3610 (close relative to the original Marburg strain). These strains have a common core genome, but differ in their content of phages and mobile elements.

All of these strains can be used as recipients for mutations from the BKE collection.
In addition, the genetic tools (plasmids, vectors, drug cassettes) routinely used in B. subtilis (including erm) are often used for studies in other Bacilli. We presented this new approach to the Bacillus community at the recent on-line "Subtillery" conference and this generated a lot of interest. We were contacted by Dr. Filho (Brazil) who wishes to apply this approach to Bacillus velezensis FZB (formerly B. amyloliquefaciens FZB). It can also be applied to members of the B. cereus group (B. thuringiensis, B. anthracis).
The manuscript describes different modifications which were performed, and it is sometimes hard to follow them, and to see what the outcome is. I would suggest to rewrite the description of the different experiments to make it clearer what was done and what is really the novel approach. ** Figure 2 presents readers with a schematic that shows the starting genome organization, and how this method can introduce a variety of modifications using selection against the erm cassette. Together with the description in the text, we think this is quite clear.
The novelty of the approach is not the type of constructions that can be made, but the idea of using a single sgRNA to target any of the nearly 4000 non-essential genes (and essential genes with a nearby erm cassette) in one of the most important model organisms (B. subtilis). This completely bypasses the need to design, synthesize, and clone the DNA encoding the sgRNA. Combined with the option of using genomic DNA as repair template, this greatly simplifies and speeds CRISPR-based editing.
In general, the manuscript is written well but very often sought is used, what might be replaced. **We thank reviewer for this point, and we noted five occurrences of the word "sought." We have removed four of these.