Genetically engineered mammalian cells have demonstrated enormous clinical potential, especially as cancer therapy. Also, immune cells equipped with a single-input reporter circuit for tumor detection can achieve higher sensitivity than traditional imaging and blood-based diagnostics in mice, thus highlighting their potential as diag- nostic devices. While promising, a major challenge in cell-based therapeutics and diagnostics is sensitivity. As such, installing signal amplification genetic circuits will greatly improve the sensitivity and performance of cell- based devices. Furthermore, most therapeutics and diagnostics rely on detecting a single biomarker, drastically limiting their specificity. Genetic circuits that can robustly and efficiently integrate multiple inputs, logically pro- cess the information, and produce the desired outputs will be required to address this challenge. One of the most important tools for genetic circuit engineering in mammalian cells is the site-specific recom- binase. An advantage of a recombinase-based gene expression circuit is its ability to amplify weak input signals. Furthermore, our lab and others have shown that recombinases are especially suitable for logic circuit engineer- ing. We have also identified many split locations within several orthogonal recombinases, which were leveraged to develop a large collection of small-molecule inducible recombinases with enhanced performance. While we have a powerful collection of recombinase-based gene circuits, there are significant gaps in our toolkit that severely limit their applicability. For instance, it remains challenging to connect recombinase to input sen- sors that display minimal basal activity and sufficient dynamic range, which degrades the performance of recom- binase-based circuits. Moreover, multi-input recombinase circuits typically require multiple orthogonal recom- binases. Since recombinase can permanently modify DNA, it has a natural memory capability, which prevents them from distinguishing sequential (e.g., A then B) vs. simultaneous appearance (A AND B) of inputs. Therefore, it would be desirable to develop circuits that can integrate various inputs with as few recombinases as possible. For this proposed work, we will develop digital enhancer circuits and self-assembled split recombinases for com- binatorial logic sensing to amplify signals and improve specificity. We will demonstrate their applications in the context of cellular diagnostic devices for ovarian cancer. To achieve our objectives, we will; Aim 1: Design a recombinase-shRNA-based digital enhancer circuit to amplify input signals Aim 2: Design a library of self-assembled split recombinases for combinatorial logic circuits Our team has all the necessary expertise to accomplish this work, as demonstrated by our published work in engineering recombinase circuits, in vivo tumor diagnostics, and tumor-targeting immune cells in vivo. Success from this proposed work will lead to a transformative platform for engineering cellular diagnostic devices. Fur- thermore, our work will also set the foundation for using engineered cells to detect other pathological conditions, such as inflammation.

Public Health Statement The genetic logic circuit is invaluable in basic research and has the potential to transform diagnostics, cell fate reprogramming, tissue engineering, and cell therapy, but their sensitivity and specificity need to be improved before they can be widely adopted. We proposed to develop tools that can enhance the sensitivity and specificity of signal detection in immune cells. Success from this work will greatly enhance our ability to design novel living devices for therapeutic and diagnostic applications.