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

Yingxiang Wang; Linfeng Cao; Meiying Bi; Sicheng Wang; Meiting Chen; Xingyu Chen; Ming Ying; Lei Huang

doi:10.3389/fchem.2021.717609

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

Front Chem. 2021 Aug 9:9:717609. doi: 10.3389/fchem.2021.717609. eCollection 2021.

Authors

Yingxiang Wang^{1

2}, Linfeng Cao^{1

2}, Meiying Bi^{1

2}, Sicheng Wang^{1

2}, Meiting Chen^{1

2}, Xingyu Chen^{1

2}, Ming Ying^{1

2}, Lei Huang^{1

2}

Affiliations

¹ Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, School of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin, China.
² Tianjin Key Laboratory of Drug Targeting and Bioimaging, School of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin, China.

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.

Keywords: Bacillus pumilus; CCR; UgRNA:Cas9; cre-box; multiple gene wobble editing.