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

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.

Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02

“…However, its low transformation efficiency and the restriction-modification system hinder the development of its CRISPR tool. Compared with traditional homologous recombination, CRISPR/Cas9 system gene editing requires shorter time and higher efficiency, and has been widely used in various animals, plants and microorganisms [ 38 – 40 ]. The CRISPR/Cas9 system in B. amyloliquefaciens was not reported for the first time until 2020 [ 19 ].…”

Self-assembled endogenous DNA nanoparticles for auto-release and expression of the eGFP gene in Bacillus subtilis

“…SMMP (300 μL) was added and incubated for 60−90 min at 37 °C in a shaking water bath (100 rev/min) to allow for the expression of the resistance and eGFP genes. The solutions were then diluted appropriately with 1 × SMM (342.3 g of sucrose, 4.64 g of maleic acid, 8.12 g of MgCl 2 ·6H 2 O, and deionized water to 1000 mL, pH 7.0,) and 0.1 mL was plated on complete medium for regeneration (CMR) (10 g of glucose, 10 g of peptone, 10 g of yeast extract, 5 g beef extract, 5 g of NaCl, 342.3 g of sucrose, 8.13 g of MgCl 2 , and deionzed water to 1000 mL, pH 7.2–7.5, and then 1.6% agar was added, before sterilization at 0.1 Mpa for 20 min) containing 25 μg/mL chloramphenicol for GpCas9 or 100 μg/mL ampicillin for GpX461 and GpY094 to select transformants 34 . The experiments were repeated in triplicate to assay colony formation per μg of plasmid DNA added in the cit Z-box loading system, while the corresponding free plasmids at the same concentration were used as controls in the transformation experiments.…”

Genome-scale cis-acting catabolite-responsive element editing confers Bacillus pumilus LG3145 plant-beneficial functions

“…Previously, we found seven target genes with mutations at their cre sites that resulted in the mutant strain exhibiting a carbon catabolite repression (CCR)-release phenotype, such as secreting a large number of secondary metabolites. 12 , 13 Here, we continue to report the editing results occurring in other regions of the genome, and used omics analysis of the mutant strain to reveal the connection between the plant-beneficial functions and cre -like site editing.…”