Advances in gene editing tools for four typical Gram-positive bacteria

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Gram-positive bacteria are crucial for synthetic biology, fermentation, and probiotics.
• Gene editing technologies are vital for engineering these bacteria.
• Challenges persist in developing efficient gene editing systems for Gram-positive bacteria due to cell wall structure and tool compatibility.

Purpose of the Study:

• To review the evolution and current status of gene editing tools in four representative Gram-positive bacteria: Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, and Corynebacterium glutamicum.
• To summarize common challenges and strategies for gene editing in these species.
• To provide recommendations for tool selection in different applications.

Main Methods:

• Review of traditional homologous recombination to modern CRISPR-Cas9, base editors, and large-fragment integration tools.
• Analysis of challenges including host repair capacity, tool compatibility, and editor limitations.
• Synthesis of strategies and recommendations for gene editing tool development and application.

Main Results:

• Gene editing tools have evolved significantly, from homologous recombination to advanced CRISPR-based systems.
• Common challenges in host repair, tool compatibility, and editor limitations were identified across the studied species.
• Specific strategies and recommendations for optimizing gene editing were proposed for different application scenarios.

Conclusions:

• Despite advancements, efficient gene editing in Gram-positive bacteria requires addressing inherent challenges.
• The findings offer a technical reference for gene editing in Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, and Corynebacterium glutamicum.
• Summarized issues may guide the development of gene editing tools for other Gram-positive bacteria.