Genome-Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1 |
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| Authors: | Dr Yukio Koizumi Prof Jun Fukushima Yayoi Kobayashi Ayumi Kadowaki Miyuki Natsui Dr Tomokazu Yamaguchi Prof Yumiko Imai Prof Toshihiro Sugiyama Prof Keiji Kuba |
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| Affiliation: | 1. Department of Biochemistry and Metabolic Science, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan;2. Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, 241–438 Kaidobata-Nishi, Shimoshinjo-Nakano, Akita, 010–0195 Japan;3. Laboratory of Regulation of Intractable Infectious Diseases, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka, 567-0085 Japan |
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| Abstract: | 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. |
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| Keywords: | CRISPR/Cas9 cytotoxicity malformin peptides squalene epoxidase |
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