Aouida Mustapha, Eid Ayman, Mahfouz Magdy M
Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia.
Biochim Open. 2016 Feb 24;3:72-75. doi: 10.1016/j.biopen.2016.02.001. eCollection 2016 Dec.
CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.
CRISPR/Cas9系统赋予古细菌和细菌物种针对入侵的外来核酸的分子免疫能力。CRISPR/Cas9系统被用于多种真核物种的基因组工程应用。在本研究中，我们证明了CRISPR/Cas9基因组工程系统在人类细胞中用于药物靶点验证的实用性。普拉地诺醇B是一种天然大环内酯类化合物，其抗肿瘤活性通过抑制前体mRNA剪接介导。为了验证普拉地诺醇B的剪接体靶点，我们使用CRSIPR/Cas9系统在HEK293T细胞的SF3B复合物亚基中引入靶向突变。我们的数据表明，SF3b1亚基的靶向诱变表现出对普拉地诺醇B更高水平的抗性。因此，我们的数据验证了普拉地诺醇B的剪接体靶点，并为使用CRISPR/Cas9系统进行药物靶点鉴定和验证提供了概念验证。
