A Golden Gate-Compatible CRISPR-Associated Transposon Tool for Multiplexed Bacterial Genome Editing

The insertion of large genetic circuits and metabolic pathways into bacterial genomes is becoming increasingly common within the field of synthetic biology due to the improved robustness and stability that come with genome integration. CRISPR-associated transposases (CASTs) enable RNA-guided DNA insertion without introducing double-stranded breaks and have been shown to function across diverse bacterial species. Here, we present an improved tool called pSPIN-GG and supporting protocols for simplified CAST-based genome engineering. The pSPIN-GG system includes Golden Gate-compatible promoter, guide, and cargo modules for simple assembly, a green fluorescent protein dropout cassette for rapid verification of guide replacement, and a set of tested sites within the Escherichia coli BL21 chromosome to enable gene dosing of genetic cargoes. These refinements support accelerated library construction, reduce assembly and screening burden, and expand the accessibility of CAST systems for multiplexed bacterial genome engineering.