肿瘤相关基因中大量变异的致病性未知，这归因于流行病学数据有限，因此将其归类为具有不确定意义的变异体（VUS）。迄今为止，乳腺癌基因2型（BRCA2）拥有最多的VUS，这促使我们开发了几种强大的功能性试验来确定它们的功能意义。本文报告了一种利用人化小鼠胚胎干细胞（mESC）系表达单一拷贝人BRCA2的CRISPR-Cas9基于高通量功能性试验的方法。作为原理的验证，我们饱和了BRCA2外显子3、18、19中编码的11个密码子和外显子13中所有可能的单核苷酸变异，并使这些变异体进行功能分类。具体来说，我们使用一个180碱基长的单链供体DNA池来生成所有可能的变异体组合。使用基于高通量测序的方法，我们显示非功能性变异体的频率显著降低，而功能性变异体在细胞池中富集。我们进一步展示了这些变异体对DNA损伤药物顺铂和奥拉帕尼的反应，使我们可以使用细胞存活和药物反应作为变异体分类的参数。利用该方法，我们已经对599个BRCA2变异体进行了分类，其中包括11个密码子中的93个单核苷酸变异（SNVs），其中28个在ClinVar中有报道。我们还对来自外显子13的252个SNVs进行了功能性分类，其中188个是功能性的，60个是非功能性的，这表明饱和基因组编辑（SGE）结合药物敏感度试验可以增强BRCA2 VUS的功能注释。 版权：本文为开放获取文章，无版权限制，任何人都可以自由复制、分发、传播、修改、建立相关内容，或以任何合法用途使用。该作品以创作共用CC0公共领域奉献的方式提供。

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.Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.