Abstract: Mycoplasma is an important zoonotic pathogen. As a cell-wall-free prokaryote, its genome is highly streamlined and is an ideal model for synthetic biology and genome minimization research. Gene editing is a core tool for analyzing the biological characteristics of mycoplasma. This paper systematically summarizes three major categories of mainstream operational technologies: Tn transposons, homologous recombination and CRISPR/Cas systems, and explains the principles of action, technical characteristics and optimization and improvement strategies of each technology;Among them, the CRISPR/Cas systems is subdivided into CRISPR/Cas9, CRISPR interference （CRISPRi）, base editing, SURE editing and other derivative tools. Different editing techniques have their respective applicable scenarios, and have been widely used in mycoplasma gene functional verification and pathogenic mechanism analysis, development of attenuated vaccines, rapid detection of pathogens, and construction of synthetic biology chassis cells and other applications: Tn transposons and CRISPR/Cas9 are suitable for pathogenic mechanism mining and vaccine development, while CRISPR-derived diagnostic techniques have unique advantages in rapid detection of pathogens. In summary, after integration and improvement, different types of gene editing tools have become the core technology system for basic research and application development of mycoplasma, which can provide key support for the prevention and control of mycoplasma diseases and the innovation of biological technology.

Key words: mycoplasma, gene editing, CRISPR/Cas, transposon, pathogenic mechanism, vaccine development, synthetic biology