Digital single cancer biomarker detection by CRISPR/Cas sensing: Towards dynamic profiling for CpG methylation quantification

Introduction
Cancer, as a disease, starts with a genetic change in the genome that results in the downregulation of genes involved in “normal” cell behavior. This genetic change can either be induced by a change in the DNA code (mutation) or epigenetic alterations, such as cytosine methylation. Hypermethylation of cytosines has shown to be a critical hallmark in many cancer cells. More specifically, the loss of expression of genes occurs about 10 times more often by hypermethylation of promotor CpG islands than by (point) mutations. For early cancer diagnostics, accurate and rapid detection of these epigenetic CpG methylation mutations involved in tumor development is crucial. Previously, we have presented an amplification-free in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. While this method showed a lot of potential in terms of specificity, selectivity and ease of use, the limit of detection presented (100 pM) was still far away from the concentrations of DNA found in liquid biopsies such as urine. Since all commonly available pre-amplification techniques used for CRISPR sensing will result in a loss of epigenetic modifications we here present a methods to lower the limit of detection of this Cas12a assay, while keeping methylation selectivity.