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  • An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.

Communications biology (2024-04-11)
Brad A Davidson, Adam X Miranda, Sarah C Reed, Riley E Bergman, Justin D J Kemp, Anvith P Reddy, Morgan V Pantone, Ethan K Fox, R Dixon Dorand, Paula J Hurley, Sarah Croessmann, Ben Ho Park
ABSTRACT

Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.

MATERIALS
Product Number
Brand
Product Description

Roche
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Tablets provided in EASYpacks
Sigma-Aldrich
TRAIL Protein, Recombinant human, Human TRAIL (TNF-Related Apoptosis Inducing Ligand), also called APO2 Ligand, is a cytotoxic protein which activates rapid apoptosis in tumor cells, but not in normal cells.