Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

Abstract

The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for Escherichia coli and Saccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery. IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in Bacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of B. subtilis strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.

MutS2 Promotes Homologous Recombination in Bacillus subtilis

“…Our initial genetic analysis resulted in the creation of a CRISPR/Cas9 genome editing system on a single, temperaturesensitive plasmid that results in markerless mutations that can be quickly introduced into many genetic backgrounds. Although this is not the first report of CRISPR/Cas9 genome editing in Bacillus subtilis (50,51), the system described here does not require an inducer for Cas9 expression and still allows for efficient removal of the plasmid editing system. We further demonstrated that the novel mutS2 phenotype depends on recA and does not depend on uvrA or recU.…”

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

“…CRISPRi holds great promise for a wide range of applications in microorganisms, including bacterial cell growth control [35], genetic screen [25, 36], synthetic biology module development [37, 38] or metabolic networks control in various microorganisms such as E. coli [24, 39, 40], mycobacteria [41], Bacillus subtilis [42], Corynebacterium glutamicum [43], Clostridium beijerinckii [44], yeast [45] and cyanobacteria [7]. In particular, a number of recent studies have exploited CRISPRi to regulate the metabolic pathways in E. coli for enhanced production of various biotechnological products including poly(3-hydroxybutyrate- co -4-hydroxybutyrate) [23], terpenoid [8], pinosylvin [46], flavonoid [47] and mevalonate [48].…”

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

“…pAW005 is a pHT01 (Nguyen, Phan, & Schumann, 2007) derivative containing a multiple cloning site (MCS) comprised of BamHI-AatII-SbfI-AscI and SpeI-NgoMIV-NotI-AsiSI-AgeI-XmaI restriction sites flanking promoter P grac.max , a modified version of P grac (i.e., promoter P01) (Phan, Nguyen, & Schumann, 2012). This cassette was inserted into SbfI/NheI-digested pAW004-2 (Westbrook et al, 2016), yielding pAW002-4. P grac.max contains the upstream promoter element (UP) of promoter P64, the −35 region of promoter P71, the −15 region of promoter P57, the native −10 region, and a T-C substitution at the +3 site (Phan et al, 2012).…”

“…Mutation of clsA or pssA, encoding phosphatidylserine synthase (PssA), significantly increased extracellular amylase production, presumably through creating a more negative membrane charge density (Cao et al, 2017). To further enhance the functional expression of SeHAS, we then altered the distribution of CL in the membrane by reducing the expression of ftsZ via CRISPR interference (CRISPRi) using our recently developed CRISPR-Cas9 toolkit for B. subtilis (Westbrook et al, 2016). The membrane CL content was increased by overexpressing pgsA and clsA, resulting in significant improvements to the HA titer and MW.…”