Abstract
BackgroundNext 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.ResultsCancer-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.ConclusionsThese 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.
Highlights
Innovative advances in DNA sequencing technology have deepened the understanding of cancer genetic abnormalities and accumulated huge volumes of data on genetic abnormalities in various human cancers [1,2,3,4]
We describe introduce the creation of the genome edited strains and their properties, and discuss about their usage including a use for the comprehensive standard for the NCC OncoPanel
Single strand oligo DNA adjacent to the target site was prepared with a length of 71–78 bp
Summary
Innovative advances in DNA sequencing technology have deepened the understanding of cancer genetic abnormalities and accumulated huge volumes of data on genetic abnormalities in various human cancers [1,2,3,4]. When the number of gene to be sequenced is limited, a portion of the clinical specimen can be preserved and used as the reference material for the specific gene, but it is difficult to prepare such standards for all the genes in the panel. The established cell line, which is considered to be closer to clinical samples than synthetic DNA has been proposed to maintain a steady supply of the homogeneous and more reliable reference material covering whole range of mutations in the cancer gene panels. Various cancer-related gene panels such as OncomineTM 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 order to create mutant standards in a defined background, we have used CRISPR/Cas genome-editing system in HEK 293 T/17 cells
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