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SRX21142706: GSM7659355: Cell line Exon13.23, DMSO, Replicate 1; Mus musculus; OTHER
1 ILLUMINA (Illumina MiSeq) run: 3.2M spots, 1.6G bases, 483.8Mb downloads

External Id: GSM7659355_r1
Submitted by: Mouse Cancer Genetics Program
Study: Saturation genome editing of 11 codons and exon 13 of BRCA2 determines pathogenicity of variants
show Abstracthide Abstract
The pathogenicity of majority of variants identified in cancer-causing genes is unknown due to limited epidemiological data, hence they are considered to be variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate that variants with partial loss of BRCA2 function are sensitive to the DNA-damaging agents, cisplatin and olaparib, allowing us to discriminate between functional and intermediate variants. We have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional, 60 non-functional and 4 intermediate variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS. Overall design: Saturation genome editing (SGE) coupled with drug sensitivity assays of BRCA2
Sample: Cell line Exon13.23, DMSO, Replicate 1
SAMN36698329 • SRS18407105 • All experiments • All runs
Organism: Mus musculus
Library:
Name: GSM7659355
Instrument: Illumina MiSeq
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Genomic DNA was extracted from the cell pellets using Zymo Genomic DNA Extraction kit DNA libraries were prepared using TruSeq Nano DNA Library Prep kit
Runs: 1 run, 3.2M spots, 1.6G bases, 483.8Mb
Run# of Spots# of BasesSizePublished
SRR254066463,216,9321.6G483.8Mb2023-08-30

ID:
28578024

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