A novel CRISPR screen identifies mediators of cfDNA release

The detection of circulating tumor DNA (ctDNA) via liquid biopsy has
become routine practice in clinical oncology. Despite its widespread use,
the majority of clinically approved tests have limited sensitivity due to
the relative paucity of ctDNA. We reasoned that understanding the
biogenesis of cell-free DNA (cfDNA) might identify opportunities to
increase DNA production from cancer cells and thereby increase the
sensitivity of clinical ctDNA testing. Here, we used a novel CRISPR screen
to identify the most relevant molecular mediators of cfDNA release and
validated these effectors using multiple human cancer and non-cancer cell
lines. Previously, apoptosis, necrosis, and extracellular vesicle release
have been posited to correlate with DNA release from cancer cells. We find
that different cell lines naturally release cfDNA in diverse amounts and
fragmentation patterns. Our results also demonstrate that the apoptotic
pathway is the major regulator of cfDNA release. Further, cfDNA fragments
released through apoptosis have traditionally been thought to be small
~167bp fragments, the size of those identified in human blood. We instead
find apoptosis can lead to cfDNA release as large >1000bp
fragments, suggesting that the cfDNA found in blood may require additional
processing towards this size. We additionally establish that TRAIL can
increase cfDNA release across the majority of cancer cell lines,
consistent with this ligand’s known proapoptotic role. These results show
that the apoptotic pathway is a major mechanism of cfDNA release, and
provide opportunities for optimizing liquid biopsies for cancer
diagnostics.