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 fragm..., CRISPR-Cas9 Screen comparing the changes in sgRNA barcode representation between DNA found in the cell-free populations to that of the genomic DNA populations, hoping to identify regulators of cfDNA release. This screen was carried out in the MCF-10A and A549 cell lines. The Brunello human CRISPR Knockout Pooled Library in the one vector format (Addgene 73179-LV) was used to individually infect these cell lines at MOIs of 0.3–0.5. After puromycin and passaging for 28 days, DNA was isolated from culture media (cfDNA) by centrifugal clearing of debris at 300g for 10 minutes and 2000g for 20 minutes followed by isolation with the Zymo Quick-DNA Urine Kit (D3061). Genomic DNA from the cells in culture was isolated using the QiaAMP DNA Blood Maxi Kit (Qiagen 51192). sgRNA barcodes were PCR amplified from these samples as well as the plasmid DNA used to create the Brunello library as outlined in the Brunello Sequencing Protocol on Addgene's Website and were then gel isolated. Sequencing was r..., , # A novel CRISPR screen identifies mediators of cfDNA Release

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Within this dataset is 9 raw sequencing files used to analyze two separate CRISPR screens in the MCF-10A and A549 cell lines within our manuscript of the same title. These screens compared the barcode representation between cell-free and genomic DNA using the MaGECK-MLE algorithm to identify barcodes that were differentially present between the two populations, which could indicate regulators of increased or decreased cell-free DNA release. In the end, our biggest hits in MCF-10A screen were in the TRAIL pathway, while in the A549 screen they were in BCL2-family genes.

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## Description of the Data and File Structure

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The Brunello human CRISPR Knockout Pooled Library in the one vector format (Addgene 73179-LV) was used to individually infect these cell lines at MOIs of 0.3-0.5. After puromycin and passaging for 28 days, DNA was isolated from culture media (cfDNA) by centrifugal clearing of debris at 300g for 10 ...