Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells

Abstract

Background: 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. Results: 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. Conclusions: 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.

A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

“…However, these samples are not appropriate in their current form for comprehensively evaluating the analytical performance of cancer panels as somatic mutations often at lower than 20% variant allele frequency (VAF). Other samples including reference standards developed for somatic mutation typically involve at most one cancer cell line and a matching normal, greatly limiting the number of relevant variants at low VAF available for evaluation [3][4][5][6]. While these reference standards are valuable and provide utility in several NGS contexts, proper comprehensive assessment of cancer panels typically requires more than 100 appropriate qualitative analytes (such as a variant detected/not detected) in each of several distinct VAF ranges, which is more than what many general and commercially available reference samples have in total.…”

Preparation of multiplexed control materials for cancer mutation analysis by genome editing in GM12878 cells

“…Paired with GM12878, they also can be used as controls for tumor‐normal sequencing. Because of its high transfection efficiency, HEK 293 are frequently used to produce genome‐edited cell lines as controls for somatic mutation testing 6,10–12 . However, HEK293 is a hypotriploid cell line which displays some cytogenetic instability and is not well‐characterized across the genome.…”

“…Feed cells may be used. Generation of mutant cells by mutation knock‐in via on homology‐directed repair is another approach, however, the efficiency is relatively low 12 . In addition, only nucleotides adjacent to protospacer adjacent motif (PAM) site can be efficiently edited with this strategy, which may exclude many clinical actional variants.…”

“…Because of its high transfection efficiency, HEK 293 are frequently used to produce genome‐edited cell lines as controls for somatic mutation testing. 6 , 10 , 11 , 12 However, HEK293 is a hypotriploid cell line which displays some cytogenetic instability and is not well‐characterized across the genome. On the contrary, GM12878 is stable, and most importantly, it is extensively characterized and serves as benchmark reference materials for NGS testing.…”

“…Generation of mutant cells by mutation knock‐in via on homology‐directed repair is another approach, however, the efficiency is relatively low. 12 In addition, only nucleotides adjacent to protospacer adjacent motif (PAM) site can be efficiently edited with this strategy, which may exclude many clinical actional variants. By using CRISPR/Cas9‐induced homology‐independent integration, we can efficiently introduce any long DNA sequence harboring design variants into any locus of chromosome.…”