CRISPR-Cas-amplified urine biomarkers for multiplexed and portable cancer diagnostics

Abstract

Synthetic biomarkers, exogenous probes that generate molecular reporters, represent an emerging paradigm in precision diagnostics with applications across infectious and noncommunicable diseases. These methods use multiplexing strategies to provide tools that are both sensitive and specific. However, the field of synthetic biomarkers has not benefited from molecular strategies such as DNA-barcoding due to the susceptibility of nucleic acids in vivo. Herein, we exploit chemically-stabilized DNAs to tag synthetic biomarkers and produce diagnostic signals via CRISPR nucleases. Our strategy capitalizes on disease-associated, protease-activated release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed biofluids. In murine cancer models, we show that these DNA-encoded nanosensors can noninvasively detect and monitor disease progression, and demonstrate that nuclease amplification can be harnessed to convert the readout to a point-of-care tool. This technique combines specificity with ease of use to offer a new platform to study human disease and guide therapeutic decisions.

Building synthetic biosensors using red blood cell proteins

“…Interesting avenues include reconstitution of a protease to modulate the ultrasound profile of encapsulated gas vesicles, 77 or to release a surface-bound synthetic biomarker that is excreted in a patient’s urine. 78, 79 There exist many approaches for choosing protein split sites, and we recently developed a computation-guided method for tuning reconstitution propensity to achieve high performance when split proteins are used in applications such as the engineered biosensors described here. 45 Finally, we validated our BRET biosensor in G1ER cells over a short period of time (< 90 min).…”