Suzuki Takayoshi, Tsukumo Yoshinori, Furihata Chie, Naito Mikihiko, Kohara Arihiro
1Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26, Tonomachi-ku, Kawasaki, 210-9501 Japan.
2JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Ibaraki City, Osaka, 567-0085 Japan.
Genes Environ. 2020 Feb 11;42:8. doi: 10.1186/s41021-020-0147-2. eCollection 2020.
Next Generation Sequencer (NGS) is a powerful tool for a high-throughput sequencing of human genome. It is important to ensure reliability and sensitivity of the sequence data for a clinical use of the NGS. Various cancer-related gene panels such as Oncomine™ or NCC OncoPanel have been developed and used for clinical studies. Because these panels contain multiple genes, it is difficult to ensure the performance of mutation detection for every gene. In addition, various platforms of NGS are developed and their cross-platform validation has become necessity. In order to create mutant standards in a defined background, we have used CRISPR/Cas9 genome-editing system in HEK 293 T/17 cells.
Cancer-related genes that are frequently used in NGS-based cancer panels were selected as the target genes. Target mutations were selected based on their frequency reported in database, and clinical significance and on the applicability of CRISPR/Cas9 by considering distance from PAM site, and off-targets. We have successfully generated 88 hetero- and homozygous mutant cell lines at the targeted sites of 36 genes representing a total of 125 mutations.
These knock-in HEK293T/17 cells can be used as the reference mutant standards with a steady and continuous supply for NGS-based cancer panel tests from the JCRB cell bank. In addition, these cell lines can provide a tool for the functional analysis of targeted mutations in cancer-related genes in the isogenic background.
新一代测序仪（NGS）是用于人类基因组高通量测序的强大工具。确保NGS临床应用中序列数据的可靠性和敏感性非常重要。已经开发了各种癌症相关基因检测板，如Oncomine™或NCC OncoPanel，并用于临床研究。由于这些检测板包含多个基因，因此难以确保每个基因的突变检测性能。此外，NGS的各种平台不断发展，其跨平台验证已成为必要。为了在特定背景下创建突变标准品，我们在HEK 293 T/17细胞中使用了CRISPR/Cas9基因组编辑系统。
选择了在基于NGS的癌症检测板中常用的癌症相关基因作为靶基因。根据数据库中报道的频率、临床意义以及通过考虑与PAM位点的距离和脱靶情况来选择CRISPR/Cas9的适用性，来选择靶突变。我们已经在36个基因的靶位点成功产生了88个杂合和纯合突变细胞系，共125个突变。
这些敲入的HEK293T/17细胞可作为参考突变标准品，由JCRB细胞库稳定持续供应，用于基于NGS的癌症检测板测试。此外，这些细胞系可为在同基因背景下对癌症相关基因中的靶突变进行功能分析提供工具。
