Gram-positive bacteria serve as important chassis microorganisms in synthetic biology, industrial fermentation, and probiotic development. The rapid advancement of gene editing technologies has provided critical technical support for the iterative construction and functional validation of engineered strains. However, due to factors such as cell wall structure, differences in genetic backgrounds, and tool compatibility, the development and editing efficiency of gene editing systems for Gram-positive bacteria still face many challenges. This review focuses on four representative Gram-positive bacterial species-Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, and Corynebacterium glutamicum-and traces the evolution and current state of their editing tools, from traditional homologous recombination to CRISPR-Cas9, base editors, and large-fragment integration tools. On this basis, we summarize the common challenges and corresponding strategies concerning host repair capacity, tool compatibility, and inherent limitations of editors in these four bacterial species, and propose recommendations for tool selection based on different application scenarios. This review aims to provide a technical reference for gene editing studies of the above-mentioned bacterial species. Although the conclusions cannot be directly extended to all Gram-positive bacteria, the common issues summarized here may inform the development of gene editing tools for other Gram-positive bacteria.
