Cell-free recombinase-integrated Boolean output system

Cell-free gene expression systems are increasingly important in fundamental research and biomanufacturing, offering a versatile platform for studying gene circuits and biocomputation. We present the cell-free recombinase-integrated Boolean output system (CRIBOS), a site-specific recombinase-based multiplex genetic circuit platform designed for cell-free environments. With CRIBOS, we built over 20 multi-input-multi-output circuits, including 2-input-2-output genetic circuits and a 2-input-4-output decoder. Combined with allosteric transcription factor (aTF)-based sensors, the circuits demonstrate multiplex environmental sensing. Moreover, utilizing paper-based CRIBOS, which demonstrates remarkable portability and stability, we present a biological memory storage logic circuit device that can preserve DNA-based biological information for over 4 months with minimal resources, energy costs, and maintenance requirements. Implementing CRIBOS not only expands the application of multiplex Boolean logic gates from cellular systems to the cell-free environment but also augments their overall versatility, opening new avenues for designing and applying sophisticated genetic circuits. A record of this paper's transparent peer review process is included in the supplemental information.