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. In order to achieve their promise, these methods reply on 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 the DNA-encoded urine biomarkers 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.

Multiplexed Profiling of Extracellular Vesicles for Biomarker Development

“…The genome editing capacity of the CRISPR/Cas system has been investigated as a novel multiplexing platform [195][196][197][198][199]. Owing to the inherent programmability of Cas proteins [200], collateral activity upon multiple 20-mer barcodes can be extended to report potentially hundreds of orthogonal codes.…”

“…Owing to the inherent programmability of Cas proteins [200], collateral activity upon multiple 20-mer barcodes can be extended to report potentially hundreds of orthogonal codes. A proof-of-concept study using CRISPR-Cas12a-mediated barcodes has been applied in the detection of urine biomarkers [197]. Massively multiplexed nucleic acid detection with Cas13-based SHERLOCK detection technology along with microfluidic chips has also been applied to both COVID-19 and other viral infections [201].…”

Interfacing Biomaterials with Synthetic T Cell Immunity

“…This opportunity to challenge current limits of detection has enabled the use of synthetic biomarkers for early disease detection in cancer and other pathological conditions. [ 54–63 ]…”

“…Multiplexed detection can be achieved by employing a library of mass‐ or DNA‐barcoded biomarkers to monitor multiple proteases simultaneously. [ 54,58,153 ] Additionally, recent advances in machine learning enable the training of diagnostic classifiers that accurately differentiate experimental groups based on multiplexed urinary outputs. Mac et al.…”

“…To increase tumor‐targeting, these biomarkers have been conjugated with tumor‐penetrating peptides or targeting ligands to enhance on‐target signals from CD8+ TILs. [ 54–56 ] Alternatively, biomarkers masked with photolabile protecting groups have been unmasked in the TME using a light trigger, facilitating selective activation by local proteases. [ 57 ] Furthermore, protease activity has been integrated in synthetic logic circuits, which could further enhance detection specificity of synthetic biomarkers.…”