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

Abstract

AbtractClinical 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.

Advances in CRISPR-based technologies for genome editing in microorganisms

“…17 In the medical field, CRISPR holds enormous promise for gene therapy and the treatment of genetic disorders. 19 It allows scientists to correct or replace faulty genes associated with diseases, offering potential cures for conditions that were once considered incurable. CRISPR facilitates the development of genetically modified crops with improved traits such as disease resistance, drought tolerance, and enhanced nutritional content.…”

Enhancing biodiversity monitoring efficiency through CRISPR-driven depletion and enrichment of aquatic environmental DNA

“…The vast diversity in organism types in an eDNA sample (e.g., vast diversity in all Bacteria, Archaea, Eukarya domains) means that DNA condition or DNA interactions within the eDNA collected can be hard to predict (Panlilio & Rice, 2021), which may impact the ability to isolate the DNA of interest. CRISPR-based screens of extracellular DNA can shed light on the mechanisms of DNA release, like those in cell free DNA in human studies (Davidson et al, 2024) and as a result inform targets for eDNA based monitoring if certain DNA fragments are more readily released than others.…”