A Biomolecular\nCircuit for Automatic Gene Regulation\nin Mammalian Cells with CRISPR Technology

We introduce a biomolecular circuit for precise control\nof gene\nexpression in mammalian cells. The circuit leverages the stochiometric\ninteraction between the artificial transcription factor VPR-dCas9\nand the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil\ndomains to boost their interaction, to maintain the expression of\na reporter protein constant across diverse experimental conditions,\nincluding fluctuations in protein degradation rates and plasmid concentrations,\nby automatically adjusting its mRNA level. This capability, known\nas robust perfect adaptation (RPA), is crucial for the stable functioning\nof biological systems and has wide-ranging implications for biotechnological\napplications. This system belongs to a class of biomolecular circuits\nnamed antithetic integral controllers, and it can be easily adapted\nto regulate any endogenous transcription factor thanks to the versatility\nof the CRISPR-Cas system. Finally, we show that RPA also holds in\ncells genomically integrated with the circuit, thus paving the way\nfor practical applications in biotechnology that require stable cell\nlines.