CRISPR/Cas9 has become a simple and powerful genome editing tool for many organisms. However, multi-round genome editing should replace single-guide RNA (sgRNA) every round, which is laborious and time-consuming. Here, we have developed a multi-round genome editing system in which genome editing and the programmed removal of the sgRNA have sequentially occurred in a growth-dependent manner in Bacillus subtilis . The system contains two plasmids, one containing a cas9 gene and the other containing two sgRNAs and a donor DNA for homology directed repair (HDR). The two sgRNAs are chromosome-targeting (sgRNA ct ) and self-targeting (sgRNA st ) under the control of a constitutive promoter and sporulation-specific promoter, respectively. In the growth phase, the sgRNA ct is transcribed and complexed with the Cas9 to edit the chromosomal target, while the sgRNA st is transcribed in the sporulation phase and complexed with the Cas9 to attack its own plasmid. Therefore, the system automatically makes the cell ready for next-round genome editing during cultivation. The system was approved through the sequential deletion of eight extracellular protease genes in the B. subtilis , suggesting that it can be used for versatile applications in multi-round genome editing.

Numerous drugs have emerged to treat various diseases, such as COVID-19, cancer, and protect human health. Approximately 40% of them are lipophilic and are used for treating diseases through various delivery routes, including skin absorption, oral administration, and injection. However, as lipophilic drugs have a low solubility in the human body, drug delivery systems (DDSs) are being actively developed to increase drug bioavailability. Liposomes, micro-sponges, and polymer-based nanoparticles have been proposed as DDS carriers for lipophilic drugs. However, their instability, cytotoxicity, and lack of targeting ability limit their commercialization. Lipid nanoparticles (LNPs) have fewer side effects, excellent biocompatibility, and high physical stability. LNPs are considered efficient vehicles of lipophilic drugs owing to their lipid-based internal structure. In addition, recent LNP studies suggest that the bioavailability of LNP can be increased through surface modifications, such as PEGylation, chitosan, and surfactant protein coating. Thus, their combinations have an abundant utilization potential in the fields of DDSs for carrying lipophilic drugs. In this review, the functions and efficiencies of various types of LNPs and surface modifications developed to optimize lipophilic drug delivery are discussed.

Bacillus strains produce various types of antibiotics, and random mutagenesis has traditionally been used to overproduce these natural metabolites. However, this method leads to the accumulation of unwanted mutations in the genome. Here, we rationally designed a single nucleotide substitution in the degU gene to generate a B. subtilis strain displaying increased plipastatin production in a foreign DNA-free manner. The mutant strain (BS1028u) showed improved antifungal activity against Pythium ultimum. Notably, pps operon deletion in BS1028u resulted in complete loss of antifungal activity, suggesting that the antifungal activity strongly depends on the expression of the pps operon. Quantitative real-time PCR and lacZ assays showed that the point mutation resulted in 2-fold increased pps operon expression, which caused the increase in antifungal activity. Likewise, commercial Bacillus strains can be improved to display higher antifungal activity by rationally designed simple modifications of their genome, rendering them more efficient biocontrol agents.