Breakthrough in Gene Editing: A Three-Plasmid CRISPR-Cas9 Platform for Bv916

Researchers at Huaiyin Institute of Technology in China have developed a novel three-plasmid CRISPR-Cas9 platform for the biocontrol agent Bacillus velezensis 916, allowing for efficient gene editing and improved biological control efficacy. This breakthrough has significant implications for sustainable agriculture, as it enables the development of Bv916 as a cell factory, integrating multiple biocontrol factors. The platform was established by incorporating optimized BvCas9 under the constitutive promoter P43, single guide RNAs (sgRNAs), and homologous recombination fragments into three thermosensitive shuttle vectors.

Key Takeaways:

• The three-plasmid CRISPR-Cas9 platform achieved a single-gene editing efficiency of 96% and simultaneous dual-gene editing efficiency of 61% within five business days.
• This platform was used to replace promoters of four non-ribosomal peptide synthetase (NRPS) gene clusters in Bv916, generating the derivative BvLSBF.
• BvLSBF showed significant increases in locillomycin (6.8-fold), surfactin (5.9-fold), bacillomycin L (10.9-fold), and fengycin (6.2-fold) production compared to Bv916.
• The antagonistic activity of BvLSBF against plant pathogens was also significantly enhanced.
• This gene editing system enables further development of Bv916 as a cell factory and integration of multiple biocontrol factors for sustainable agriculture.
• The research was conducted by Kecheng Luo and his team at Huaiyin Institute of Technology, in collaboration with other researchers.
• The study was published in the journal Applied and Environmental Microbiology.

Statistics:

• 96% single-gene editing efficiency
• 61% simultaneous dual-gene editing efficiency
• 6.8-fold increase in locillomycin production
• 5.9-fold increase in surfactin production
• 10.9-fold increase in bacillomycin L production
• 6.2-fold increase in fengycin production
• 5 business days for gene editing and editing

Sources:

• A three-plasmid-containing CRISPR-Cas9 platform to engineer Bacillus velezensis 916 as an efficient biocontrol agent. Applied and Environmental Microbiology, 2025.
• Amer Soc Microbiology, 1752 N St NW, Washington, DC 20036-2904, USA (American Society for Microbiology - www.asm.org; Applied and Environmental Microbiology - aem.asm.org)
• Kecheng Luo, Jiangsu Provincial Key Construction Laboratory of Probiotics Preparation, Huaiyin Institute of Technology, Huaian, People's Republic of China
• Lian Li, Shuangyu Zhang, Xiaohua Wang, Sihan Wang, Xuehui Liu, Shanshan Zang, Yuan Liu, Changyong Zhou, and Chuping Luo