The release of fetoplacental cell-free DNA (cfDNA) into the maternal bloodstream opened up new avenues towards noninvasive prenatal testing (NIPT) for aneuploidies, hereditary DNA mutations and other pregnancy-related developmental disorders. Increasingly, cfDNA catches interest for its noninvasive screening value in other areas as well, including oncology. Although there are indications that cfDNA fragmentation is a non-random process, the etiology and different structural aspects of cfDNA are still not well known. The emerging field of cfDNA fragmentomics investigates the existence of tissue and disease specific cfDNA signatures and the chemistry and biology underlying the fragmentation process. This review sheds light on recent developments in cfDNA fragmentomics and illustrates their significance in NIPT improvement and beyond.We discuss aspects of fragment size distributions, epigenetic correlations and putatively enriched cfDNA fragment-end compositions. Combinatorial fragmentomic efforts have provided more insights into the roles of different enzymes that contribute to the fragmentation process in the tissue of origin and in the bloodstream. Altogether, these studies revealed multiple fragmentomic-related biomarkers that can be used to make noninvasive screening and other types of clinical use of cfDNA more robust, by raising its distinctive capacities. This includes multiple complementary approaches to determine the fetal fraction, a key determinant in NIPT. Furthermore, these developments translate to a better understanding of the encountered cfDNA patterns and will catalyze the expansion of screening possibilities in NIPT and beyond

Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis , exhibits indiscriminate ssDNase activity in trans , which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called “hotspots.” In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.