A novel CRISPR screen identifies mediators of cfDNA release
Data files
Mar 20, 2024 version files 17.72 GB
Abstract
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.
README: 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 -> cfDNA and plasmid DNA -> 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.
Methods
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 -> cfDNA and plasmid DNA -> genomic DNA arms of each screen to identify hits that were potential regulators of cfDNA release.