Abstract 815: Using CRISPR/Cas9 to Generate Isogenic Cell Lines and Reference Standards for Applications in Cancer Diagnostics
Cancer research(2017)
摘要
Abstract We are entering an era of precision medicine, in which there are significant initiatives to employ companion diagnostics and molecular profiling to inform clinical decisions in therapeutic treatment. In order for these initiatives to work as effectively as possible, there is a need for molecular reference materials that can help facilitate precision assay development. Cell-line based reference standards are ideal for such applications because they represent a reproducible and renewable source of control materials that can be employed for assay development, quality control, or lead compound discovery. With this in mind, we have used the CRISPR/Cas9 platform to engineer series of isogenic cell lines that feature 51 recurrent mutations in the MAPK signaling pathway. These cell lines contain footprint-free, homozygous mutations in the kinase domains of the EGFR, KRAS, and BRAF genes in HCT116 and RKO cancer cell lines, with 32 mutations in EGFR, 12 mutations in KRAS, and 7 mutations in BRAF. The mutations include precise deletions, point mutations and small fragment insertions, and these engineered cell lines have been further employed for the development of derivative reference materials, including genomic DNA (gDNA) and FFPE cell line blocks. Such materials can be employed as positive and negative controls in diverse assay platforms, including next generation sequencing (NGS), quantitative PCR (qPCR), in situ hybridization (ISH), and others. We present quality control data for these reference materials, which demonstrate excellent consistency and uniformity, as well as examples of the application of these materials in molecular assay validation. We conclude that using CRISPR/Cas9 technology to generate molecular reference standards is a very efficient and precise method in generating footprint-free, biorelevant mutations for companion diagnostic assays. Citation Format: Huanyu Jin, Xin Cheng, Nga Nguyen, Qi Zheng, Mi Shi, Andrew Hilmer, Yuan Yuan, Ruby Yanru Chen-Tsai, Ling-Jie Kong. Using CRISPR/Cas9 to generate isogenic cell lines and reference standards for applications in cancer diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 815. doi:10.1158/1538-7445.AM2017-815
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