ACS Synthetic Biology

CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics

The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for an easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of-care detection. Here, we established a

Engineering Native-like GPCR Antigens Enables Discovery of an LPA2-Blocking Antibody for Ovarian Cancer

G-protein-coupled receptors (GPCRs) are high-value therapeutic targets, yet antibody discovery remains limited by difficulties in preparing antigens that preserve native conformations. Here, we engineered a native-like, full-length human LPA2 antigen by combining N-terminal P9* fused with amphipathic poly-γ-glutamate (APG) stabilization, affording an antigen suitable for the selection of antibodies with therapeutic efficacy. By screening a large synthetic human scFv library, we isolated an antagonistic antibody against LPA2 that bound LPA2 selectively over LPA1 (EC

One-Pot Isothermal Linear Amplification and Cas12a-Based Nucleic Acid Detection

CRISPR-based nucleic acid diagnostics are a promising class of point-of-care tools that could dramatically improve healthcare outcomes for millions worldwide. However, these diagnostics require nucleic acid preamplification, an additional step that complicates deployment to low resource settings. Here, we developed CATNAP (Cas

Synthetic Circuit-Driven Expression of Heterologous Enzymes for Disease Detection

The integration of nanotechnology and synthetic biology could lay the framework for new classes of engineered biosensors that produce amplified readouts of disease states. As a proof-of-concept demonstration of this vision, here we present an engineered gene circuit that, in response to cancer-associated transcriptional deregulation, expresses heterologous enzyme biomarkers whose activity can be measured by nanoparticle sensors that generate amplified detection readouts. Specifically, we designed an AND-gate gene circuit that integrates the activity of two ovarian cancer-specific synthetic promoters to drive the expression of a heterologous protein output, secreted Tobacco Etch Virus (TEV) protease, exclusively from within tumor cells. Nanoparticle probes were engineered to carry a TEV-specific peptide substrate in order to measure the activity of the circuit-generated enzyme to yield amplified detection signals measurable in the urine or blood. We applied our integrated sense-and-respond system in a mouse model of disseminated ovarian cancer, where we demonstrated measurement of circuit-specific TEV protease activity both