The CRISPRaTOR: a biomolecular circuit for Automatic Gene Regulation in Mammalian Cells with CRISPR technology

We introduce the CRISPRaTOR, a biomolecular circuit for precise control of gene expression in mammalian cells. The CRISPRaTOR leverages the stochiometric interaction between the artificial transcription factor VPR-dCas9, and the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil domains to boost their interaction, to maintain the expression of a reporter protein constant across diverse experimental conditions, including fluctuations in protein degradation rates and plasmid concentrations, by automatically adjusting its mRNA level. This capability, known as Robust Perfect Adaptation (RPA), is crucial for the stable functioning of biological systems and has wide-ranging implications for biotechnological applications. The CRISPRaTOR belongs to a class of biomolecular circuits named antithetic integral controllers, and it can be easily adapted to regulate any endogenous transcription factor thanks to the versatility of CRISPR-Cas system. Finally, we show that RPA holds also in cells genomically integrated with the CRISPRaTOR, thus paving the way for practical applications in biotechnology that require stable cell lines.