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.

The development of DNA delivery techniques is critical to promote the wider use of deoxyribonucleic acids as cellular transporters. The present study aimed to develop a type of DNA nanoparticle (citZ-box) to automatically load and release cargo. The restriction enzyme can cleave citZ-boxes at pro-designed sites, and the enhanced green fluorescent protein gene (eGFP) can be delivered into the B. subtilis protoplasts by them. The process of eGFP expression is recorded using a confocal microscope over 4 h. Here, multiscaffold and multimodular designs are used for citZ-box assembly with a DAEDALUS module, DX_cage_design and rem (edge_length, 21), to ensure the structure was predicted as B-type DNA. Finally the citZ-box is estimated to be a 50.7 nm cube. The 3D structure of the citZ-box particle is detected to be approximately 50.3 ± 0.3 nm. DNA nanoparticles prepared as citZ-boxes have great potential as drug carriers with automatic loading and releasing abilities.

Piriformospora indica , a plant root-colonizing basidiomycete fungus, exhibits strong growth-promoting activity in symbiosis with a broad range of plants. Here, we report the potential of P. indica to improve growth, yield, and disease resistance in wheat in the field. In the present study, P. indica successfully colonized wheat through chlamydospores and formed dense mycelial networks that covered roots. Plants subjected to the seed soaking (SS) treatment with P. indica chlamydospore suspensions enhanced tillering 2.28-fold compared to the non-inoculated wheat in the tillering stage. In addition, P. indica colonization promoted vegetative growth significantly during the three-leaf, tillering, and jointing stages. Moreover, the P. indica -SS-treatment enhanced wheat yield by 16.37 ± 1.63%, by increasing grains per ear and panicle weight and decreased damage to wheat shoot and root architecture markedly, with high field control effects against Fusarium pseudograminearum (81.59 ± 1.32%), Bipolaris sorokiniana (82.19 ± 1.59%), and Rhizoctonia cerealis (75.98 ± 1.36%). Most of the primary metabolites, such as amino acids, nucleotides, and lipids, involved in vegetative reproduction were increased in P. indica -SS-treatment plants, whereas secondary metabolites, such as terpenoids, polyketides, and alkaloids, decreased following P. indica inoculation. The up-regulated processes of protein, carbohydrate, and lipid metabolism indicated that P. indica colonization increased growth, yield, and disease resistance via the acceleration of plant primary metabolism. In conclusion, P. indica improved morphological, physiological, and metabolic substance levels, and further promoted its growth, yield, and disease resistance in wheat.