A portable Cas6f-based system for multiplex translational repression in bacteria

Engineered small RNAs (sRNAs) enable programmable gene knockdowns and support metabolic engineering and multiplex regulation in model bacteria. Still, precise, tunable, and multiplex gene repression remains a challenge in synthetic biology. Common tools can impose genetic burden, depend on host RNA factors, or do not transfer well across species. Here we present MORTISE (Multiplex, ORthogonal Translation Interference SystEm), a compact Cas6f-based platform for programmable translational repression in Gram-negative bacteria. The system functions without host Hfq or RNases and operates robustly in Escherichia coli and Pseudomonas putida. We demonstrate repression in both species using chromosomal reporter assays, with performance improving when guide and target transcription are matched and when the translation initiation region is targeted. Single-promoter multiplexing enables simultaneous knockdowns and a cloning toolbox facilitates assembly of up to nine guides in a single step. Finally, MORTISE is leveraged to boost malonyl-coenzyme A-dependent production in P. putida, supporting pathway balancing.