Abstract
CRISPR-associated Cas9 endonuclease (CRISPR/Cas9) systems are widely used to introduce precise mutations, such as knocking in/out at targeted genomic sites. Herein, we successfully disrupted the transcription of multiple genes in Bacillus pumilus LG3145 using a series of unspecific guide RNAs (gRNAs) and UgRNA:Cas9 system-assisted cre-box editing. The bases used as gRNAs shared 30–70% similarity with a consensus sequence, a cis-acting element (cre-box) mediating carbon catabolite repression (CCR) of many genes in Bacillus. This triggers trans-crRNA:Cas9 complex wobble cleavage up/downstream of cre sites in the promoters of multiple genes (up to 7), as confirmed by Sanger sequencing and next-generation sequencing (NGS). LG3145 displayed an obvious CCR release phenotype, including numerous secondary metabolites released into the culture broth, ∼ 1.67 g/L white flocculent protein, pigment overflow causing orange-coloured broth (absorbance = 309 nm), polysaccharide capsules appearing outside cells, improved sugar tolerance, and a two-fold increase in cell density. We assessed the relationship between carbon catabolite pathways and phenotype changes caused by unspecific UgRNA-directed cre site wobble editing. We propose a novel strategy for editing consensus targets at operator sequences that mediates transcriptional regulation in bacteria.
Highlights
Bacillus pumilus, a Gram-positive soil bacterium, is considered a potential host strain for the production of chemicals, heterologous proteins and antimicrobial materials (Westers et al, 2004; Su et al, 2020)
Chloromycetin (25 μg/ml) was added to the CMR media used for selecting transformants harbouring the UgRNA:Cas9 system
The genomes of the mutants were determined by next-generation sequencing (NGS) and the results indicate that most of the mutation sites occurred around the seven target fragments, but some mutation sites occurred at other sites
Summary
A Gram-positive soil bacterium, is considered a potential host strain for the production of chemicals, heterologous proteins and antimicrobial materials (Westers et al, 2004; Su et al, 2020). Wobble Editing by Unspecific CRISPR/Cas in B. pumilus proximal cis-acting conserved sequence that negatively regulates multiple genes in Bacillus species; the histidine phosphocarrier protein Hpr, an intermediate of the phosphoenolpyruvate:sugar transport system (PTS); and the catabolite control protein CcpA, a trans-acting factor that binds cre-box through Hpr-Ser-p to control target gene transcription (Peng et al, 2020; Langa et al, 2021; Neira et al, 2021). We wished to manipulate this trigger in Bacillus pumilus to disrupt the transcriptional regulation of multiple genes simultaneously to overcome CCR limitations, enhance sugar resistance, and improve secretion. The off-target effects may be utilised to edit the consensus sequence at multiple-gene loci such as cre-boxes. Three 20 nt oligonucleotides sharing 30–70% similarity with target sites were synthesised to serve as single gRNAs involved in targeting seven genes (listed in Supplementary Table S1)
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