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A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair.
Nucleic Acids Research ( IF 14.9 ) Pub Date : 2020-09-17 , DOI: 10.1093/nar/gkaa748
Kohei Nishimura 1, 2 , Ryotaro Yamada 3, 4 , Shinya Hagihara 3, 4 , Rie Iwasaki 3 , Naoyuki Uchida 3, 5 , Takumi Kamura 2 , Koji Takahashi 2, 3 , Keiko U Torii 3, 6, 7 , Tatsuo Fukagawa 1
Affiliation  

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

中文翻译:

具有工程化的生长素-TIR1对的超灵敏的生长素诱导型德隆系统。

生长素诱导型德隆(AID)系统通过应用天然生长素IAA能够快速耗尽细胞内的靶蛋白。AID系统可用于研究必需蛋白质的生理功能。但是,该系统通常需要高剂量的生长素才能在脊椎动物细胞中实现有效耗竭。在这里,我们描述了一种超灵敏的AID系统,该系统结合了合成的生长素衍生物5-Ad-IAA及其高亲和力结合伴侣OsTIR1 F74A。超灵敏的AID系统使鸡DT40细胞中的AID诱导剂浓度降低了1000倍以上。为了将此系统应用到包括包含多组染色体的癌细胞在内的各种哺乳动物细胞系中,我们利用了一步法,其中基于CRISPR / Cas9的基因敲除与pAID质粒的插入相结合。单步方法与超灵敏AID系统相结合,使我们能够轻松,快速地在各种脊椎动物细胞系中生成基于AID的条件性基因敲除细胞。我们改进的方法结合了超灵敏的AID系统和单步方法,为阐明必需基因的作用提供了强大的工具。
更新日期:2020-10-11
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