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.
