CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIa Intron-19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells

Results indicate that CRISPR/Cas9 gene editing of a suboptimal E19/I19 5' splice site in the TOP2a gene results in circumvention of acquired drug resistance to etoposide and other TOP2-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 thereby decreasing formation of a truncated TOP2a/90 isoform and increasing expression of full-length TOP2a/170 in these resistant cells. Overall design: An etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2a/170, expresses high levels of a novel C-terminal truncated TOP2a isoform (90 kDa, TOP2a/90). TOP2a/90, the translation product of a TOP2a mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2a/170 and is a resistance determinant through a dominant negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2a-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was utilized to make changes (GAG//GTAAAC?GAG//GTAAGT) in the TOP2a gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS).

研究结果表明，对TOP2a基因中功能欠佳的E19/I19 5'剪接位点（5' splice site, 5' SS）实施CRISPR/Cas9基因编辑，可通过增强第19内含子的剪接切除效率，减少截短型TOP2a/90异构体的生成，并提高耐药细胞中全长型TOP2a/170的表达水平，从而规避克隆化K562细胞系中对依托泊苷及其他靶向TOP2的药物产生的获得性耐药。
实验整体设计：一株因TOP2a/170表达水平降低而获得依托泊苷耐药的K562克隆化亚株K/VP.5，会高表达一种新型C端截短型TOP2a异构体（分子量90 kDa，即TOP2a/90）。该TOP2a/90是保留未剪接第19内含子（I19）的TOP2a mRNA的翻译产物，可与TOP2a/170形成异二聚体，并通过对药物活性产生显性负效应成为耐药性决定因子。我们提出假说：通过基因组编辑增强I19的剪接切除，可作为一种可行策略以规避获得性TOP2a介导的药物耐药。为在K/VP.5细胞中增强I19的剪接切除效率，本研究利用CRISPR/Cas9技术对TOP2a基因中功能欠佳的外显子19/内含子19 5'剪接位点（E19/I19 5' SS）进行了序列改造，突变位点为GAG//GTAAAC→GAG//GTAAGT。