Department of Pediatrics, School of Medicine, University of Yamanashi, Chuo, Japan.
Innovation Medical Research Institute, Research and Development Center for Precision Medicine, University of Tsukuba, Ibaraki, Japan.
Sci Rep. 2018 Jul 2;8(1):9966. doi: 10.1038/s41598-018-27767-6.
In many cancers, somatic mutations confer tumorigenesis and drug-resistance. The recently established clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a potentially elegant approach to functionally evaluate mutations in cancers. To reproduce mutations by homologous recombination (HR), the HR pathway must be functional, but DNA damage repair is frequently impaired in cancers. Imatinib is a tyrosine kinase inhibitor for BCR-ABL1 in Philadelphia chromosome-positive (Ph+) leukemia, and development of resistance due to kinase domain mutation is an important issue. We attempted to introduce the T315I gatekeeper mutation into three Ph+ myeloid leukemia cell lines with a seemingly functional HR pathway due to resistance to the inhibitor for poly (ADP) ribose polymerase1. Imatinib-resistant sublines were efficiently developed by the CRISPR/Cas9 system after short-term selection with imatinib; resulting sublines acquired the T315I mutation after HR. Thus, the usefulness of CRISPR/Cas9 system for functional analysis of somatic mutations in cancers was demonstrated.
在许多癌症中，体细胞突变赋予肿瘤发生和耐药性。最近建立的成簇规律间隔短回文重复序列 (CRISPR)/Cas9 系统是一种功能评估癌症突变的潜在优雅方法。为了通过同源重组 (HR) 产生突变，HR 途径必须是功能性的，但在癌症中，DNA 损伤修复经常受损。伊马替尼是费城染色体阳性 (Ph+) 白血病中 BCR-ABL1 的酪氨酸激酶抑制剂，由于激酶结构域突变而产生耐药性是一个重要问题。我们试图将 T315I 看门突变引入三个似乎具有功能性 HR 途径的 Ph+髓系白血病细胞系，因为它们对聚 (ADP) 核糖聚合酶 1 的抑制剂有耐药性。在使用伊马替尼进行短期选择后，CRISPR/Cas9 系统有效地开发了伊马替尼耐药亚系;HR 后获得了 T315I 突变。因此，证明了 CRISPR/Cas9 系统在癌症体细胞突变的功能分析中的有用性。
