DOI: 10.5061/dryad.k0p2ngfd2
title: A novel CRISPR screen identifies mediators of cfDNA release

description: 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 &gt;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.
description: 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 run on a NovaSeq at the Vanderbilt
 "VANTAGE" Genomics Core, and is deposited in raw fq.gz format
 for re-analysis here. Files were analyzed using MaGECK-VISPR with the
 MaGECK-MLE algorithm. Comparisons of beta scores (fold-change) were made
 between plasmid DNA -&gt; cfDNA and plasmid DNA -&gt; genomic DNA
 arms of each screen to identify hits that were potential regulators of
 cfDNA release. 
description: # A novel CRISPR screen identifies mediators of cfDNA Release --- 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. --- ##
 Description of the Data and File Structure --- 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 gel isolated. Sequencing was run on a
 NovaSeq at the Vanderbilt "VANTAGE" Genomics Core, and are
 deposited in raw fq.gz format for re-analysis here. Afterwards, files were
 analyzed using MaGECK-VISPR with the MaGECK-MLE alorithm. Comparisons of
 beta scores (fold-change) were made between plasmid DNA -&gt; cfDNA and
 plasmid DNA -&gt; genomic DNA arms of each screen to identify hits which
 were potential regulators of cfDNA release. Genes were considered
 potential hits when the beta scores produced in each arm of the screen
 were sufficiently diferent and were significantly selected in opposing or
 differential directions as outlined by p-values generated through
 MaGECK-MLE. Files used in our analysis as are follows: MCF-10A Plasmid DNA
 Files: 4269-BD-1-TTGAGTTA-TCTTTCCC_S22_R1_001.fastq.gz
 4269-BD-2-TTGAGTTA-TCTTTCCC_S166_R1_001.fastq.gz MCF-10A genomic DNA
 Files: 4269-BD-3-GGCTCACG-TCTTTCCC_S23_R1_001.fastq.gz
 4269-BD-4-GGCTCACG-TCTTTCCC_S167_R1_001.fastq.gz MCF-10A cell-free DNA
 Files: 4269-BD-5-CCTCCATA-TCTTTCCC_S24_R1_001.fastq.gz
 4269-BD-6-CCTCCATA-TCTTTCCC_S168_R1_001.fastq.gz A549 Plasmid DNA File:
 8229-BD-1_S1_L005_R1_001.fastq.gz A549 genomic DNA File:
 8229-BD-5_S1_L005_R2_001.fastq.gz A549 cell-free DNA File:
 8229-BD-4_S1_L005_R1_001.fastq.gz --- ## Sharing/Access Information ---
 This data is accessible only through Dryad at the moment, and was
 originally generated entirely within the Ben Ho Park Lab at Vanderbilt
 University Medical Center with aid from the Vanderbilt VANTAGE Genomics
 Core. Outside of this depository, please contact the Ben Ho Park Lab for
 additional information.