Scaling up Genetic Circuits in Mammalian Cells: A U1-snRNA-based Platform Enables Mammalian Cells to Compute the Bitwise Inversion of the Square Root of a Number

Scaling up genetic circuits in mammalian cells can lead to a new class of therapies. As a result of my research as PhD candidate on exploring ways and engineering tools to scale up genetic circuits, I engineered and validated in HEK293FT cells a genetic circuit that allows those cells to compute the bitwise inversion of the square root of a number. To date, this circuit which has four inputs and two outputs is the most sophisticated genetically encoded circuit ever expressed in mammalian cells. The core processing module of the circuit is a novel miRNA-based NOT gate based on a platform that uses the U1 snRNA. We have called this platform "u.P.R.O.C.E.S.S.O.R." (U-gene-based Platform, RNAi-regulated Only, Compactly Employing Small Shuttle-miRNAs, Operates (through) RNA), which does not use any transcriptional regulators or exogenous proteins, which can cause dangerous immune responses. The design of this sophisticated logic circuit was found by executing an algorithm, which I developed, for the exhaustive search of logic circuits designs (with 4 inputs and 2 outputs). The solution tested in HEK293FT cells required just four transcriptional units and about 10kb of DNA. Furthermore, I have engineered a trans-activated gRNA (for CAS9) and a trans-activated miRNA to sense abundant nuclear RNAs by the use of the toehold-mediated strand displacement reaction. The transactivated miRNA also does not use any transcriptional regulators or exogenous proteins and, like the miRNA-based NOT gate, has a DNA footprint small enough to fit in a AAV virus.Ph.D.S.M