SARGE: Serine Integrase-Assisted Rapid Genome Integration through Cassette Exchange in Escherichia coli.

Serine integrases enable precise genome engineering but remain underused for iterative genome integration because current workflows are often labor-intensive and difficult to scale. Here, we report SARGE, a serine integrase-assisted rapid genome integration platform, that enables iterative insertion of large DNA fragments into the Escherichia coli genome through cassette exchange. By combining the orthogonal serine integrases PhiC31 and Bxb1 with rational donor plasmid design, SARGE supports rapid, programmable integration cycles without the need for resistance marker excision between rounds. To simplify the identification of correct recombinants, we incorporated a dual-fluorescence reporter system based on sfGFP and mKate and developed a green-red screening strategy for direct, naked-eye colony selection. This visual screen identified the desired recombinants with 100% accuracy among the colonies tested. SARGE achieved cassette exchange efficiencies of up to 95% and maintained efficiencies of approximately 90% for cargoes as large as 10 kb. Together, these features substantially streamline iterative genome integration and establish SARGE as a robust and accessible platform for genome engineering and synthetic biology in E. coli, with potential for extension to other genetically tractable microbial hosts.