基于CRISPR/Cas9-sgRNA全基因组敲除文库筛选系统鉴定DON致炎症反应关键毒效应靶标及其机制研究

DON是自然界中一类不可避免的B类单端孢霉烯族毒素，动物长期暴露于该毒素中将严重抑制动物生长，给畜牧业带来巨大经济损失。前期我们已经证实，DON引起严重的炎症反应是DON所致器官损伤、体重下降、生长迟缓的重要原因。据此我们推测“DON所致炎症反应的关键毒效应靶标的发现是缓解或降低DON毒性的关键环节”。本课题首先在前期建立的DON导致细胞炎症反应模型基础上，应用CRISPR/Cas9-sgRNA全基因组敲除文库的高效筛选系统，深入挖掘DON所致细胞损伤的关键靶标；其次，以关键靶标为切入点，结合CRISPR/Cas9基因编辑、高通量测序、蛋白超表达、DARTS和CETSA等技术确定DON与靶标的直接或间接作用；再进而揭示靶标相关信号通路及其毒作用机制，全面系统阐释DON导致细胞炎症反应的分子机制，推动高效、安全的DON新型拮抗剂的研发。

DON is an inevitable class B toxin in nature, and long term exposure to the toxin will severely inhibit animal growth and bring huge economic losses to animal husbandry. Our previous studies have identified that severe inflammatory reaction caused by DON plays important roles in the organ damage, weight loss and growth retardation. Therefore, we speculate that "the discovery of the key toxic target of the inflammatory reaction caused by DON is the key point to alleviate or reduce the toxicity of DON". Based on the previous model of cellular inflammatory reaction caused by DON, the high-efficiency screening system with CRISPR /Cas9-sgRNA whole genome knockout library was used to investigate the key targets of cell damage induced by DON. Then, taking the target as the entry point, the direct or indirect interactions between DON and the targets were investigated by using CRISPR /Cas9 gene editing, high-throughput sequencing analysis, protein overexpression, DARTS and CETSA, and other technologies. Additionally, the targets related signal pathways and toxic mechanism were revealed to comprehensively and systematically explain the molecular mechanism of cellular inflammatory reaction caused by DON, which will promote the research and development of the new high-efficiency and safety antagonists of DON.