Rapid, simple, and ultrasensitive quantitation of KRAS ctDNA at the point of care using CRISPR/Cas amplification and digital resolution biosensor microscopy

摘要 虽然越来越多的药物可用于治疗癌症中的特定分子异常，但治疗 现在可以通过检测血浆中特定的基因组循环肿瘤DNA(CtDNA)来预测疗效。 虽然下一代测序(NGS)可以提供对基因组肿瘤变体的全面读数， 可能提供生物和临床疗效洞察，其成本、复杂性和样本到答案的时间框架是 与频繁的、常规的、护理点诊断不兼容。同时，目前可用的基于实验室的 用于对用于液体活检的血浆中经策略选择的ctDNA生物标志物进行量化的方法缺乏敏感性， 临床需求所需的多路复用和工作流程简单性。一种基因组液体活检，可以迅速 在办公室访问的时间范围内在临床环境中进行，提供了一个引人注目的替代方案来确定 特定碱基的循环核酸分子的存在、不存在和浓度变化 序列代表驱动癌症相关细胞过程的突变。这样的方法将会 能够在最早的时间进行治疗选择，同时促进更频繁的缓解 监控。为了解决目前技术上的差距，我们寻求开发并严格验证一种新的检测方法 名为“Activate，Cave，Capture，and Count”(AC3)的方法，它结合了两个创新元素。首先，我们 应用最近展示的具有数字分辨率的光子晶体(PC)生物传感器显微技术 能够量化表面捕获的金纳米颗粒(AuNP)标签。其次，我们利用CRISPR/CA 具有靶标特异的引导RNA探针的系统，该探针选择性地激活连接AuNPs的单链DNA系链的切割 到表面，为每个ctDNA分子产生许多释放的AuNPs。发布的AuNPs随后 在PC生物传感器上捕获，在那里它们被数字计数。我们的“先放大后数字化”战略提供了 令人信服的基于数字聚合酶链式反应技术的替代方案，同时还规避了 热放大、微滴分配和基于荧光的检测。基于初步的 结果对于癌症相关ctDNA的检测，AC3提供了50ZM的检测限和一种测量 突变等位基因频率为0.001%。重要的是，AC3利用了一种小巧且廉价(约7K美元)的检测 乐器。在这个项目中，我们将应用AC3来表征血浆ctdna生物标志物，横跨六个 使用添加的校准标准和库中的人血浆中的突变和表征性能 样本。我们将严格表征AC3相对于液滴的灵敏度、选择性和重复性 数字聚合酶链式反应(DdPCR)。我们设想AC3将作为基于组织的NGS的补充，应用于常规的初始癌症 筛选治疗选择，监测治疗效果，并作为缓解监测工具。 与替代药物相比，AC3固有的更高的敏感性提供了进行早期癌症治疗的机会 检测，集成更高级别的多路复用，并减少血浆体积要求。

Abstract While a growing arsenal of drugs is available to treat specific molecular abnormalities across cancers, therapy effectiveness can now be predicted by detecting specific genomic circulating tumor DNA (ctDNA) in plasma. While next-generation sequencing (NGS) can provide a comprehensive readout of genomic tumor variants that may provide biological and clinical efficacy insights, its cost, complexity, and sample-to-answer timeframe are not compatible with frequent, routine, point of care diagnostics. Meanwhile, currently available laboratory-based methods for quantifying strategically-selected ctDNA biomarkers in plasma for liquid biopsy lack sensitivity, multiplexing, and workflow simplicity required for clinical needs. A genomic liquid biopsy that can be rapidly performed in a clinical setting in the timeframe of an office visit offers a compelling alternative for identifying the presence, absence, and concentration changes in circulating nucleic acid molecules whose specific base sequences represent mutations that drive cancer-associated cellular processes. Such an approach would enable therapy selection to be performed at the earliest time while facilitating more frequent remission monitoring. To address the gaps in current technology, we seek to develop and rigorously validate a novel assay method called “Activate, Cleave, Capture, and Count” (AC3) that combines two innovative elements. First, we apply a recently-demonstrated photonic crystal (PC) biosensor microscopy technology with digital resolution capability for quantifying surface-captured gold nanoparticle (AuNP) tags. Second, we utilize the CRISPR/Cas system with target-specific guide RNA probes that selectively activate cleavage of ssDNA tethers linking AuNPs to a surface, generating many released AuNPs for each ctDNA molecule. The released AuNPs are subsequently captured on a PC biosensor, where they are digitally counted. Our ”amplify-then-digitize” strategy offers a compelling alternative to digital PCR-based technologies while also circumventing the limitations inherent with thermal amplification, microdroplet partitioning, and fluorescence-based detection. Based upon preliminary results for the detection of cancer-associated ctDNA, AC3 offers a detection limit of 50 zM and a measurement of mutant allele frequency of <0.001%. Importantly, AC3 utilizes a small and inexpensive (~ $7K) detection instrument. In this project, we will apply AC3 for characterization of plasma ctDNA biomarkers across six mutations and characterize performance using spiked-in calibration standards, and in banked human plasma samples. We will rigorously characterize the sensitivity, selectivity, and repeatability of AC3 compared to droplet digital PCR (ddPCR). We envision AC3 as a complement to tissue-based NGS, applied to routine initial cancer screening for therapy selection, monitoring the effects of treatments, and as a remission monitoring tool. Compared with alternatives, the inherently greater sensitivity of AC3 offers opportunities to perform earlier cancer detection, integrate higher levels of multiplexing, and reduce plasma volume requirements.