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CRISPR/Cas9 Editing of the Bacillus subtilis Genome   

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Original research article

A brief version of this protocol appeared in:
Journal of Bacteriology
Jan 2017

Abstract

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with unparalleled facility. CRISPR/Cas9 has allowed for genome edits to be more precise, while also increasing the efficiency of transferring mutations into a variety of genetic backgrounds. As a result, the advantages are realized in tractable organisms and organisms that have been refractory to genetic manipulation. Here, we describe our method for editing the genome of the bacterium Bacillus subtilis. Our method is highly efficient, resulting in precise, markerless mutations. Further, after generating the editing plasmid, the mutation can be quickly introduced into several genetic backgrounds, greatly increasing the speed with which genetic analyses may be performed.

Keywords: Genome editing, CRISPR, Cas9, Bacillus subtilis, Gene deletion, Point mutation

Background

Bacillus subtilis is a highly tractable, Gram-positive bacterium. It is amenable to genetic studies, using a variety of vectors to quickly and efficiently introduce mutations by homologous recombination. Although there are many different methods to introduce mutations in B. subtilis, each method has its limitations. A simple and straightforward method to make a mutation in B. subtilis is gene disruption, wherein a plasmid is integrated within a gene of interest (Vagner et al., 1998). The major limitations include: 1) the potential for polar effects by disrupting an operon; 2) introduction and retention of foreign DNA; 3) once an antibiotic resistance cassette is used, the researcher has to use a different cassette if a given mutation is to be studied in the context of other mutations; and 4) the method is limited to targeting an entire gene and cannot yield more precise point mutations. Another method employed in B. subtilis genetic studies is allelic replacement, wherein a gene of interest is replaced with an antibiotic resistance cassette (Guerout-Fleury et al., 1996). Although polar effects should be reduced by simply replacing one gene with another, this method still suffers from several limitations described above. Recently, a gene deletion library was constructed which allows for the removal of the antibiotic resistance cassette (strains are available from the Bacillus genetic stock center). As a result, researchers can use the same method for many mutations because the resistance cassette is removed after each allelic replacement. The method is an improvement, although it is still limited to gene deletions and cannot be used for point mutations. Finally, there are two methods to introduce markerless mutations in B. subtilis including point mutations. One method utilizes the upp gene as a counter-selectable marker (Fabret et al., 2002), and the other uses a plasmid called pMad (Arnaud et al., 2004) or its derivative pMiniMad which allows for mutation integration after removal of the integrating vector (Patrick and Kearns, 2008). Although these methods can introduce precise point mutations, our experience (making four gene deletions and inserting gfp at one genetic locus) using the latter method (Arnaud et al., 2004; Patrick and Kearns, 2008) is that it is quite time consuming with a success rate that is not very high (on average, about 12% success). Although we do not have experience with the upp counter selection method, the authors engineered a GGA→GAC change in the lexA gene and reported the intended change in sequence for three out of four screened isolates with the incorrect isolate yielding multiple mutations in the targeted lexA gene (Fabret et al., 2002). A major drawback, though, is that the method requires deletion of the endogenous upp gene in B. subtilis, which requires that the Δupp strain be used as the new ‘wild-type’ control. Therefore, although the methods described above work, we were in search of a genome editing method with a higher efficiency that also required less time at the bench. These criteria prompted us to adapt a CRISPR/Cas9 genome editing system (Jiang et al., 2013) to B. subtilis (Burby and Simmons, 2017). CRISPR/Cas9 can be used to introduce a variety of mutations including gene deletions, fusions, and even point mutations (Sternberg and Doudna, 2015). Further, by constructing the editing system on a single broad host-range plasmid with a temperature sensitive origin of replication, all vector DNA introduced during the procedure can easily be removed. Success rates have proven to be much higher for point mutations and small, gene-sized deletions (often over 80% success, but 100% success is not atypical), reducing the number of isolates that need to be screened. Although this method solves many of the limitations of the contemporary methods, our CRISPR/Cas9 genome editing system is limited by the requirement of a proto-spacer adjacent motif or PAM sequence (NGG in our system), and the requirement of two cloning steps. Nonetheless, the ability to make a variety of markerless mutations, coupled with the rapidity with which the mutations can be transferred to different genetic backgrounds still provides a significant improvement over current methods for genome editing in B. subtilis. The system we have developed may also be applicable to other Gram-positive bacteria with little or no manipulation of the DNA reagents described herein.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Burby, P. E. and Simmons, L. A. (2017). CRISPR/Cas9 Editing of the Bacillus subtilis Genome. Bio-protocol 7(8): e2272. DOI: 10.21769/BioProtoc.2272.
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