The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as 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 the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, 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 and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

中文翻译：

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BRCA2 11 个密码子和外显子 13 的饱和基因组编辑结合化疗药物反应，可准确确定变异的致病性。

由于流行病学数据有限，癌症相关基因中发现的大量变异的致病性未知，导致它们被归类为意义不确定的变异（VUS）。迄今为止，乳腺癌基因 2 (BRCA2) 的 VUS 数量最多，因此需要开发几种强大的功能测定法来确定其功能意义。在这里，我们报告了使用表达人类 BRCA2 单拷贝的人源化小鼠胚胎干细胞 (mESC) 系进行基于 CRISPR-Cas9 的高通量功能测定。作为原理验证，我们饱和了 BRCA2 外显子 3、18、19 和外显子 13 中所有可能的单核苷酸变体编码的 11 个密码子，并对这些变体进行多重分析以进行功能分类。具体来说，我们使用 180 聚体单链供体 DNA 池来生成所有可能的变体组合。使用基于高通量测序的方法，我们发现非功能性变异的频率显着下降，而功能性变异在细胞库中富集。我们进一步证明了这些变异体对 DNA 损伤剂顺铂和奥拉帕尼的反应，使我们能够使用细胞存活和药物反应作为变异体分类的参数。使用这种方法，我们对 599 个 BRCA2 变体进行了分类，包括跨 11 个密码子的 93 个单核苷酸变体 (SNV)，其中 28 个在 ClinVar 中报告。我们还将外显子 13 的 252 个 SNV 进行功能分类，分为 188 个功能变体和 60 个非功能变体，证明饱和基因组编辑 (SGE) 与药物敏感性测定相结合可以增强 BRCA2 VUS 的功能注释。