Programmed gRNA Removal System for CRISPR-Cas9-Mediated Multi-Round Genome Editing in Bacillus subtilis

Abstract

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.

Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains

“…The recombinant strains were grown in LB broth (containing 25 μg/ml kanamycin) at 30°C (220 rpm) for 3 h. The culture was added 0.4% D-mannose to induce Cas9 protein expression for 10 h. After induction of bacteria subculture (Altenbuchner, 2016 ), the plasmid-cured colonies were PCR-screened by diluting and plating the bacterial culture medium onto LB agar (containing 25 μg/ml kanamycin), and incubating it at 30°C overnight.…”

“…To evaluate the sterilization efficiency of the two plasmids (pJART and pJ16ST), the constructed strains pJ16ST/A16PI2 and pJART/A16PI2 were cultured at 28°C for 3 h, 0.4% D-mannose was added (Altenbuchner, 2016 ), and the culturing was continued for 12 and 24 h, respectively. Strains cultured without D-mannose were the control group.…”

“…It has been successfully applied to mice, pigs, zebrafish, arabidopsis, sorghum, nematodes, yeast, Escherichia coli and many other animals, plants and microorganisms, and has become a genome editing tool widely used in various fields of biology and medicine (Citorik et al, 2014 ). pJOE8999 is a CRISPR/Cas9 single plasmid system constructed by V. Müller, who used it for B. subtilis genome editing to construct mutants quickly and efficiently (Altenbuchner, 2016 ).…”

Synthetic Biology Tools for Genome and Transcriptome Engineering of Solventogenic Clostridium

“…Furthermore, a recent advancement, such as phage serine integrase mediated sitespecific genome engineering technique for C. ljungdahlii could be extended to other Clostridium species (Huang et al, 2019). The synthetic biology techniques that have been applied in other microorganisms may also be adopted to solventogenic clostridia in the near future: CRISPR associated site-specific insertion of transposons and base editing techniques (Ronda et al, 2015;Zhang et al, 2016;Lim and Choi, 2019;Strecker et al, 2019b). Utilization of improved clostridia strains could be a starting point for development of an industrial scale, commercially viable bio-based fuel and chemical production using Clostridium sp.…”

Cytosine Base Editor-Mediated Multiplex Genome Editing to Accelerate Discovery of Novel Antibiotics in Bacillus subtilis and Paenibacillus polymyxa

“…Many genome editing approaches have been developed for microorganism (Suzuki et al, 2005;Karstentischer et al, 2006;Jeong et al, 2015), and currently the most valued technology is CRISPR/ Cas-mediated homologous recombination, which has high accuracy and efficiency (So et al, 2017;Tian et al, 2017). Nonetheless, multiplex genome editing using the CRISPR/Cas systems remains to struggle owing to a low recombination efficiency of multiple double-strand breaks (DSBs) generated by Cas9 nucleases and the excess time required for iterative editing (Jiang et al, 2015;Adiego-Pérez et al, 2019;Lim and Choi, 2019). A recently developed tool called the cytosine base editor (CBE) involves the fusion of Cas9 nickase (nCas9; D10A mutation) and cytidine deaminase, which enables precise base editing in yeast or mammalian cells without DSBs (Komor et al, 2016(Komor et al, , 2017.…”