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