Engineering biocomputers in mammalian cells
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Endowing cells with enhanced decision-making capacities is essential for creating smarter therapeutics and for dissecting phenotypes. Implementation of synthetic gene circuits affords a means for enhanced cellular control and genetic processing; however, genetic circuits for mammalian cells often require extensive fine-tuning to perform as intended. Here, a robust, general, and scalable system, called 'Boolean logic and arithmetic through DNA excision' (BLADE) is presented that is used to engineer genetic circuits with multiple inputs and outputs in mammalian cells with minimal optimization. The reliability of BLADE arises from its reliance on site-specific recombinases that regulate genes under the control of a single promoter that integrates circuit signals on a single transcriptional layer. Using BLADE, >100 circuits were tested in human embryonic kidney and Jurkat T cells and a quantitative metric was used to evaluate their performance. The circuits include a 3-input, two-output full adder; a 6-input, one-output Boolean logic look-up table; and circuits that incorporate CRISPR–Cas9 to regulate endogenous genes. Moreover, a large library of over 15 small-molecule, light and temperature-inducible recombinases has been established for fine-tuned control. BLADE enables execution of sophisticated cellular computation in mammalian cells, with applications in cell and tissue engineering.