CRISPR-based diagnostics for circulating cell-free DNA: a paradigm shift in precision oncology

Abstract

Circulating cell-free DNA (cfDNA) has been established as a minimally invasive liquid biopsy biomarker with utility in the diagnosis of cancer, monitoring of treatment response, and detection of minimal residual disease. The clinical utility of cfDNA is currently constrained by the low abundance of circulating cfDNA fragments, high fragmentation rates, and short half-life, making it technically challenging to detect in a patient sample. Current molecular approaches for cfDNA detection, including ddPCR and NGS, are time-intensive, expensive, and unsuitable for low-resource settings and point-of-care testing. The CRISPR-Cas system offers a novel and operationally simple approach to cfDNA detection by being single nucleotide specific and compatible with isothermal and amplification-free workflows. In this review, we discuss CRISPR-based assays for cfDNA, beginning from Cas9 enrichment-type assays to promising collateral cleavage platforms employing Cas12a and Cas13a that have countered traditional bottlenecks concerning diagnostic testing. We also provide a comparative analysis of the emerging platforms for key cancer mutations with a discussion around translational scope, including implications from CRISPR-based diagnostic patents. The convergence of sensitivity, speed, multiplexing, and microfluidic integration of CRISPR diagnostics will undoubtedly constitute a next-generation approach for cfDNA analysis, presenting a great promise in impacting precision oncology and increasing access to cancer diagnostics across low-resource settings.