The development of effective biomarkers for diagnosing complex diseases like cancer is crucial for improving patient outcomes. While endogenous biomarkers like proteins or nucleic acids exist, their limited abundance or stability in circulation poses challenges. Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, offer a promising solution. These sensors amplify diagnostic signals, improving signal-to-noise ratios. However, clinical applications require highly multiplexed readouts to classify diverse disease states. DNA barcoding is a powerful multiplexing tool in analytical chemistry but its susceptibility to nucleases *in vivo* limits its utility in synthetic biomarker applications. The inherent instability of unmodified nucleic acids in circulation poses a significant hurdle for *in vivo* disease sensing using DNA multiplexing. This paper describes a novel approach that overcomes these challenges using chemically stabilized DNA barcodes and CRISPR-Cas nucleases for a highly sensitive and specific readout in urine samples, thereby enabling non-invasive, multiplexed detection of various cancers.

The existing literature highlights the need for improved biomarkers for cancer diagnosis, particularly those that are non-invasive and can provide multiplexed readouts. Endogenous biomarkers have limitations in terms of abundance and stability. Existing engineered synthetic biomarkers have shown promise but lack the high-throughput capabilities necessary for complex disease classification. DNA barcoding is a powerful technique, but the instability of unmodified nucleic acids *in vivo* has hindered its use in this context. The authors review the challenges associated with existing approaches and the rationale for their novel strategy, which leverages chemically modified DNA barcodes and CRISPR-Cas-mediated detection for a highly multiplexed and sensitive system.

The researchers engineered DNA-encoded synthetic urine biomarkers (DNA-SUBs) that combine several key technologies. First, chemically stabilized DNA, specifically phosphorothioate-modified DNA, is used to create the barcodes, which are resistant to degradation *in vivo*. These barcodes are attached to a nanocarrier, either a biological nanobody for targeted delivery or a synthetic polymer for broader application. The nanocarrier incorporates a protease substrate; when exposed to disease-specific proteases in the tumor microenvironment, the substrate is cleaved, releasing the DNA barcodes. These barcodes are then concentrated in the urine through renal filtration. The detection system employs the CRISPR-Cas12a enzyme. The released DNA barcodes activate Cas12a, which in turn cleaves a fluorescent reporter molecule or a reporter on a lateral flow assay strip, producing a detectable signal. For multiplexed detection, different barcodes are used to represent different sensors targeting specific proteases, allowing for simultaneous monitoring of multiple disease hallmarks. The study employed several in vivo models: human prostate cancer (PCa) xenografts in nude mice, a syngeneic model of metastatic murine colorectal cancer (CRC) in immunocompetent BALB/c mice, and an immunocompetent, autochthonous mouse model of lung adenocarcinoma. The researchers performed several experiments, including in vitro characterization of Cas12a activation by various DNA lengths and modifications, in vivo biodistribution studies of the nanosensors, and measurements of urinary DNA barcodes using fluorescence kinetics or lateral flow assays. They also implemented a massively multiplexed microfluidic platform for parallel detection of a large number of barcodes. Statistical analysis methods such as t-tests, ANOVA, and ROC curves were used to assess the performance of the biomarkers.

The study demonstrated the successful development and validation of a novel multiplexed platform for non-invasive cancer detection using urine samples. The chemically modified DNA barcodes showed significantly improved stability *in vivo* compared to unmodified DNA, allowing for detection in unprocessed urine. The use of CRISPR-Cas12a provided a highly sensitive and specific detection method, with subnanomolar sensitivity achieved in both fluorescent and paper-based readouts. A singleplex system utilizing a tumor-targeting nanobody successfully detected prostate cancer in a xenograft model, showing a clear difference in Cas12a activation between tumor-bearing and healthy mice. Multiplexed DNA-SUBs effectively monitored the progression of colorectal cancer lung metastases in a syngeneic model, with specific barcodes showing increased activation over time. The multiplexed system also successfully distinguished between different cancer types within the same tissue microenvironment (lung) by detecting distinct patterns of protease activity. A massively multiplexed microfluidic platform demonstrated the ability to simultaneously detect 44 different DNA barcodes in human urine, showcasing the scalability of the technology. The study showed that combinations of two to three barcodes were sufficient to distinguish disease states in isogenic murine models while suggesting that a much larger multiplexed barcode set would be needed for clinical translation. The system's ability to distinguish between different cancer types highlights the potential for personalized diagnostics based on specific protease profiles.

The findings of this study address the critical need for non-invasive, highly multiplexed cancer diagnostics. The use of chemically stabilized DNA barcodes and CRISPR-Cas12a offers a significant improvement over existing methods by combining high sensitivity, specificity, and multiplexing capabilities with the simplicity and affordability of a point-of-care test. The ability to detect different cancer types based on distinct protease activity patterns emphasizes the potential for personalized medicine, tailoring diagnostic and treatment approaches based on individual patient profiles. The massively multiplexed microfluidic platform further enhances the potential of the technology for high-throughput screening and complex disease classification. This work offers a significant advance in cancer diagnostics and has potential to be adapted for the detection of other diseases.

This research presents a highly sensitive and multiplexed platform for non-invasive cancer detection in urine, leveraging chemically stabilized DNA barcodes and CRISPR-Cas12a. The platform successfully differentiated cancer types and stages in murine models, paving the way for potential clinical translation. The development of a massively multiplexed microfluidic platform extends the technology’s applicability to more complex scenarios. Future studies should focus on clinical validation in human subjects to further refine the diagnostic capabilities and investigate the broader applicability to a wider range of diseases.

The study was conducted in murine models, which may not fully recapitulate the complexity of human disease. The translation to human subjects requires further investigation, including evaluation of sensor performance in diverse patient populations with various comorbidities. While a 5-plex DNA-SUB panel differentiated diseases in the mouse models, larger panels may be necessary for robust disease classification in humans with more diverse protease expression profiles.