This invention discloses a highly efficient and scarless method for editing the
genome of
myxobacteria and its applications. This invention combines the ISDra2 TnpB cleavage
system of *Gastrococcus radiodurans* with the MxRedET
homologous recombination system of
myxobacteria to form a one-step scarless editing platform (MxDIRECT). ISDra2 is responsible for precisely locating and cleaving the target
DNA, and MxRedET then efficiently integrates or replaces fragments at the cleavage site, enabling
gene knockout,
insertion, and replacement operations. This method increases the editing efficiency of
myxobacteria from 1% to 100%, shortens the cycle from 3-6 months to 5-7 days, and allows for one-step two-site scarless editing through a two-site design. A single cleavage can knock out up to approximately 80kb of
DNA, while two cleavages can knock out up to 200kb; and single
nucleotide substitutions can be performed at any location, achieving precise base editing. This technology provides an efficient and precise tool for modifying the
functional genes and metabolic pathways of myxobacterial
chassis cells, significantly advancing
synthetic biology research.