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 4000 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 non-essential 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 co-transformed 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.

Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system

“…Various high efficiency, simultaneous multilocus integration methods based on two-plasmid CRISPR-Cas9 systems have been implemented in E. coli [42,43], yeasts [44,45], and fungi [46,47], making them suitable host cells to be used as a chassis in the field of synthetic biology. However, although the emergence of the CRISPR/ Cas9 system has led to numerous new applications and developments in B. subtilis [29,30,[48][49][50][51], efficient methods for simultaneous gene insertion in B. subtilis…”

Recent advances of the biological and biomedical applications of CRISPR/Cas systems

“…Traditional methods utilized for genetic engineering including ZFNs and TALENs are labor intensive and time consuming in comparison with CRISPR/Cas systems [21]. CRISPR/ Cas-system-mediated genetic modification has achieved great success in modifying genome of a broad spectrum of bacterial strains and fungus including but not limited to vancomycin-resistant Enterococcus faecium, Mycobacterium tuberculosis, Lactobacillus plantarum, Corynebacterium glutamicum, Lactobacillus casei, Bacillus subtilis, Clostridium beijerinckii Saccharomyces cerevisiae and Streptomyces [22][23][24][25]. The genome engineering of microbacteria and fungus enables the modification of specific metabolic pathways, which usually gives rise to production of high Fig.…”

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages

“…Ever since the CRISPR tools were established in Bacillus , they have been simplified, improved and combined with existing resources to facilitate genome manipulations (Koo et al, 2017 ; Sachla et al, 2021 ). An example worth highlighting is the engineering of the BKE collection of strains by recruiting the CRISPR‐Cas9 system without designing specific sgRNAs.…”

“…An example worth highlighting is the engineering of the BKE collection of strains by recruiting the CRISPR‐Cas9 system without designing specific sgRNAs. The BKE collection comprises nearly 4000 B. subtilis single‐gene knockout strains with each gene replaced by the erythromycin ( erm )‐resistant gene (Sachla et al, 2021 ). By employing pJOE8999‐derived plasmids with an anti‐erm sgRNA, genome editing can be targeted to any BKE strain with the repair templates integrated into plasmids or either as PCR products or genomic DNA.…”

“…By employing pJOE8999‐derived plasmids with an anti‐erm sgRNA, genome editing can be targeted to any BKE strain with the repair templates integrated into plasmids or either as PCR products or genomic DNA. Efficient gene replacements, site‐specific mutations, intergenic region modification and the introduction of gene–reporter fusions have all been demonstrated (Sachla et al, 2021 ). This illustrates progress, but the research on the CRISPR‐based tools in Bacillus species is still at an early stage and far from the optimal state.…”