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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.

Wobble Editing of Cre-box by Unspecific CRISPR/Cas9 Causes CCR Release and Phenotypic Changes in Bacillus pumilus