Picking Up the Pieces: Advances in CRISPR Technology for Accessible Liquid Biopsy Diagnostics

Liquid biopsies based on cell-free DNA (cfDNA) offer a minimally invasive route to genetic diagnostics, for example in prenatal screening, oncology, and infectious disease. However, their clinical potential is constrained by low target abundance, biological complexity, and practical implementation challenges. This thesis explores complementary strategies to improve the performance and accessibility of liquid biopsy–based genetic testing by combining insights into cfDNA fragmentation biology with advances in CRISPR-based diagnostics. A central theme of this work is that cfDNA is not a random byproduct of cell death, but a structured and information-rich substrate. Non-random cfDNA fragmentation patterns can be leveraged to enhance diagnostic signal, and robust assay design requires understanding of the mechanisms underlying these patterns. For controlled experimental interrogation of cfDNA fragmentation, we developed an isogenic cell line model as reproducible framework for systematic study of nuclease-driven fragmentation. This thesis investigates CRISPR-based diagnostics (CRISPRdx) as a detection method for liquid biopsies. Cas12a-based assays were developed and optimized through crRNA multiplexing and trans-cleavage reporter engineering, resulting in improved assay performance. The feasibility and limitations of amplification-free detection revealed kinetic constraints that currently impede single-molecule quantification in low-level clinical samples. The use of cell line–derived cfDNA enabled efficient benchmarking of specificity, sensitivity, and single-nucleotide discrimination without reliance on scarce clinical material. Together, this work illustrates how integrating cfDNA biology with rational CRISPR assay design can advance sensitive, adaptable, and potentially point-of-care–compatible genetic diagnostics. By "picking up the pieces" of fragmented DNA, this thesis bridges fundamental molecular insight and translational diagnostic innovation.