Method for identifying essential gene using CRISPR mediated knock-out and Next-generation sequencing

Abstract

The CRISPR-Cas9 system is a powerful tool for targeted gene mutagenesis. Targeting genes with CRISPR-Cas9 can make the genes knock-out by flameshift mutation and cause them to lose their function. The CRISPR-mediated gene knock-out is widely applied for making knock-out cell line and studying gene function. Here, I report a method to identify essential gene for cell viability and pathogenic mutation by CRISPR-Cas9 mediated gene knock-out. Previous experiments resulted in the failure of the BRCA1, tumor suppressor gene, to make a knocked out cell line, so it was selected as a target gene to determine whether it was an essential gene or not. CRISPR-Cas9 performed genome editing in the human cell and making insertions or deletions at the target genes. The mutation rate of target genes was measured by Next-generation sequencing (NGS) at constant intervals and was confirmed to changes. These results enable to identify whether BRCA1 is essential gene for cell viability. Furthermore, it was estimated that pathogenic mutations of BRCA1 would occur at the locations of the exons that had been deactivated.|크리스퍼 유전자 가위는 타겟 유전자를 돌연변이를 유발시킬 수 있는 강력한 유전자 편집 도구로 사용되고 있습니다. 크리스퍼 유전자 가위를 이용하여 타겟 유전자에 프레임 시프트 돌연변이를 만들어 녹아웃을 일으킬 수 있습니다. 이러한 크리스퍼를 이용한 유전자 녹아웃은 녹아웃 세포주를 제작하거나 유전자의 기능을 연구하는데 널리 사용되고 있습니다. 우리는 유전자 녹아웃을 활용하여 세포 생존에 필수적인 유전자인지 확인하고 병원성 돌연변이를 알아내는 방법에 대한 연구를 보고 합니다. 이전에 진행했던 실험에서 종양 억제 유전자인 BRCA1이 녹아웃 된 세포주를 얻지 못했던 결과가 있었고 그 원인이 BRCA1이 필수 유전자인 것으로 추측하게 되었습니다. 따라서 BRCA1이 필수 유전자가 맞는 지 확인하기 위한 타겟 유전자로 선정하게 되었습니다. 선정 된 타겟 유전자에 크리스퍼 유전자 가위를 통해 인간 세포 내에서 타겟 유전자의 게놈 편집을 진행하였고 유전자의 삽입 또는 결실을 일으켰습니다. 차세대 시퀀싱을 통해 타겟 유전자의 돌연변이율을 일정한 날짜의 간격을 가지고 측정하였고 변화를 확인 할 수 있었습니다. 이 결과를 가지고 BRCA1이 필수 유전자인지 확인 할 수 있었고 녹아웃 된 엑손의 위치에서 BRCA1의 병원성 돌연변이가 유발되는 것으로 추정할 수 있었습니다.; The CRISPR-Cas9 system is a powerful tool for targeted gene mutagenesis. Targeting genes with CRISPR-Cas9 can make the genes knock-out by flameshift mutation and cause them to lose their function. The CRISPR-mediated gene knock-out is widely applied for making knock-out cell line and studying gene function. Here, I report a method to identify essential gene for cell viability and pathogenic mutation by CRISPR-Cas9 mediated gene knock-out. Previous experiments resulted in the failure of the BRCA1, tumor suppressor gene, to make a knocked out cell line, so it was selected as a target gene to determine whether it was an essential gene or not. CRISPR-Cas9 performed genome editing in the human cell and making insertions or deletions at the target genes. The mutation rate of target genes was measured by Next-generation sequencing (NGS) at constant intervals and was confirmed to changes. These results enable to identify whether BRCA1 is essential gene for cell viability. Furthermore, it was estimated that pathogenic mutations of BRCA1 would occur at the locations of the exons that had been deactivated.