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.

Keywords: CRISPR/Cas9; Cancer gene panel; Genome editing; Mutant standard; Next generation sequencer.

Fig. 1

Results of T7E1 assay. After transfection of expression vectors pRGEN_U6_SG, pRGEN-Cas9-CMV and ssODN corresponding to the target genes (KRAS, NRAS, PIK3CA, PTEN, BRAF and TP53), genomic DNA was isolated from the transfected cells. The target region was amplified by PCA with corresponding primers. After denature and re-annealing of the PCR product, it was digested by T7 endonuclease I (T7E1) which cut mismatched DNA fragments. Cleaved band suggests an introduction of mutations by ssODN

Fig. 2

Confirmation of the TP53 743 G > A mutation by Sanger sequencing. Existence of the targeted knock-in mutation was checked for each isolated clones by the Sanger sequencing after PCR amplification of the target region. Successful examples for TP53 743 G > A mutation are shown. Both homo and hetero knock-in mutants were obtained