Gene insertion and transcriptional regulation of Escherichia coli based on CRISPR-associated transposases

Synthetic biology aims to construct robust microbial cell factories for sustainable biomanufacturing. A major obstacle lies in the difficulty of efficiently integrating large polycistronic expression cassettes into the genome and flexibly regulating gene expression. Here, a programmable tool MUSCULAR-CAST was developed based on type IF CRISPR-associated transposases (CAST) system Tn6677. Using MUSCULAR-CAST, we have achieved efficient genomic integration of various size polycistronic expression cassettes (1-10K). Among these, a human milk oligosaccharide 3-fucosyllactose (3-FL) chassis strain was successfully constructed, with similar yields and better growth compared with that of plasmid expression strain, and a plasmid-free cutinase recombinant expression strain was constructed, with enzyme activity higher than that of plasmid containing strain. Meanwhile, we developed a gene repression tool Tn-CRISPRi based on the targeting module of MUSCULAR-CAST, which achieved high single-gene repression across diverse PAM sequences and near-complete dual-gene suppression (98.6-99.8%). Applying Tn-CRISPRi to repress 17 genes competing with 3-FL biosynthesis or nonessential for growth revealed that knockdown of osmoregulated periplasmic glucans biosynthesis protein H (mdoH) and motility protein A (motA) increased 3-FL titers by 2.79- and 4.4-fold, respectively. This study establishes MUSCULAR-CAST and Tn-CRISPRi as efficient tools for genomic integration and transcriptional regulation, providing a scalable framework for advanced chassis strain engineering in synthetic biology.