Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be ‘read out’ via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.

合成生物标记物、生物工程传感器可在患病微环境中生成分子报告基因，代表了精确诊断的新兴范例。尽管 DNA 条形码可用作多重分析工具，但它们在体内对核酸酶的敏感性限制了其实用性。在这里，我们利用化学稳定的核酸来多重合成生物标志物，并在生物流体中产生可以通过 CRISPR 核酸酶“读出”的诊断信号。该策略依靠微环境内肽酶触发核酸条形码的释放，并在未处理的尿液中进行无聚合酶扩增、CRISPR-Cas介导的条形码检测。我们的数据表明，DNA 编码的纳米传感器可以非侵入性地检测和区分移植和本土小鼠癌症模型中的疾病状态。我们还证明，可以利用 CRISPR-Cas 扩增将读数转换为即时纸质诊断工具。最后，我们采用微流体平台进行密集多重、CRISPR 介导的 DNA 条形码读出，可以快速评估复杂的人类疾病并指导治疗决策。