Genetic circuits engineered for mammalian cells often require extensive fine-tuning to perform their intended functions. To overcome this problem, we present a generalizable biocomputing platform that can engineer genetic circuits which function in human cells with minimal optimization. We used our Boolean Logic and Arithmetic through DNA Excision (BLADE) platform to build more than 100 multi-input-multi-output circuits. We devised a quantitative metric to evaluate the performance of the circuits in human embryonic kidney and Jurkat T cells. Of 113 circuits analysed, 109 functioned (96.5%) with the correct specified behavior without any optimization. We used our platform to build a three-input, two-output Full Adder and six-input, one-output Boolean Logic Look Up Table. We also used BLADE to design circuits with temporal small molecule-mediated inducible control and circuits that incorporate CRISPR/Cas9 to regulate endogenous mammalian genes.

Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

Site-specific DNA recombinases are some of the most powerful genome engineering tools in biology. Chemical and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, the availability of inducible recombinases is scarce due to the challenge of engineering high performance systems with low basal activity and sufficient dynamic range. This limitation constrains the sophistication of genetic circuits and animal models that can be created. To expand the number of available inducible recombinases, here we present a library of >20 orthogonal split recombinases that can be inducibly dimerized and activated by various small molecules, light, and temperature in mammalian cells and mice. Furthermore, we have engineered inducible split Cre systems with better performance than existing inducible Cre systems. Using our orthogonal inducible recombinases, we created a "genetic switchboard" that can independently regulate the expression of 3 different cytokines in the same cell. To demonstrate novel capability with our split recombinases, we created a tripartite inducible Flp and a 4-Input AND gate. We have performed extensive quantitative characterization of the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs in terms of signal-to-noise ratio (SNR). To facilitate sharing of this set of reagents, we have deposited our library to Addgene. This library thus significantly expands capabilities for precise and multiplexed mammalian gene expression control.

Cell-based therapies that employ engineered T cells—including those modified to express chimeric antigen receptors (CARs)—to target cancer cells have demonstrated promising responses in clinical trials. However, engineered T cell responses must be regulated to prevent severe side effects such as cytokine storms and off-target responses. Here we present a class of recombinase-based gene circuits that will enable inducible, one-time state switching in adoptive T cell therapy using an FDA-approved drug, creating a generalizable platform that can be used to control when and how strongly a gene is expressed. These circuits exhibit memory such that induced T cells will maintain any changes made even when the drug inducer is removed. This memory feature avoids prolonged drug inducer exposure, thus reducing the complexity and potential side effect associated with the drug inducer. We have utilized these circuits to control the expression of an anti-Her2-CAR, demonstrating the ability of these circuits to regulate CAR expression and T cell activity. We envision this platform can be extended to regulate other genes involved in T cell behavior for applications in various adoptive T cell therapies.

Cell-based therapies that employ engineered T cells-including the expression of chimeric antigen receptors (CARs)-to target cancer cells have demonstrated promising responses in clinical trials. However, engineered T cell responses must be regulated to prevent severe side effects such as cytokine storms and off-target responses. Here we present a class of recombinase-based gene circuits that will enable inducible switching between two states of adoptive T cell therapy using an FDA-approved drug, creating a generalizable platform that can be used to control when and how strongly a gene is expressed. These circuits exhibit memory such that induced T cells will maintain any changes made even when the drug inducer is removed. This memory feature avoids prolonged drug inducer exposure, thus reducing the complexity and potential side effect associated with the drug inducer. We have utilized these circuits to control the expression of an anti-Her2-CAR, demonstrating the ability of these circuits to regulate CAR expression and T cell activity. We envision this platform can be extended to regulate other genes in T cell behavior for various adoptive T cell therapies.