Genome‐Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1
Genome‐Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1
复制标题
基因组规模 CRISPR/Cas9 筛选揭示角鲨烯环氧酶是马尔福明 A1 细胞毒性的易感因子
DOI：
10.1002/cbic.201800769
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发表时间：
2019
期刊：
影响因子：
3.2
通讯作者：
Kuba Keiji
中科院分区：
文献类型：
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作者：
Koizumi Yukio;Fukushima Jun;Kobayashi Yayoi;Kadowaki Ayumi;Natsui Miyuki;Yamaguchi Tomokazu;Imai Yumiko;Sugiyama Toshihiro;Kuba Keiji
Maltumor A1（MA 1）是一种真菌产生的环状五肽。 MA 1对植物表现出致畸性、纤维蛋白溶解增强活性和对哺乳动物细胞的细胞毒性。为了阐明MA 1的细胞毒性机制，我们通过使用基于CRISPR/Cas9的全基因组敲除文库来筛选参与MA 1在单核细胞样U937细胞中的细胞毒性的基因。通过阳性选择对MA 1处理具有抗性的细胞进行筛选，并通过高通量测序分析整合到MA 1抗性细胞中的单向导RNA（sgRNA）。作为评估在MA 1抗性细胞中富集的sgRNA的结果，编码角鲨烯环氧酶的SQLE被鉴定为候选基因AQULE耗尽的U937细胞在MA 1存在下是存活的，并且角鲨烯环氧酶抑制剂赋予野生型细胞MA 1抗性。这些结果表明角鲨烯环氧酶与MA 1的细胞毒性有关。这一发现代表了对MA 1用于治疗缺血性疾病的应用的新见解。
Malformin A1 (MA1) is a fungus‐produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis‐enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using a CRISPR/Cas9‐based genome‐wide knockout library. Screening was performed by positive selection for cells that were resistant to MA1 treatment, and single guide RNAs (sgRNAs) integrated into MA1‐resistant cells were analyzed by high‐throughput sequencing. As a result of the evaluation of sgRNAs that were enriched in MA1‐resistant cells,SQLE, which encodes squalene epoxidase, was identified as a candidate gene.SQLE‐depleted U937 cells were viable in the presence of MA1, and squalene epoxidase inhibitor conferred MA1 resistance to wild‐type cells. These results indicate that squalene epoxidase is implicated in the cytotoxicity of MA1. This finding represents a new insight into applications of MA1 for treating ischemic diseases.