title: Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.
abstract: Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.
authors.affiliation: Center of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Toyoaki
authors.initials: T
authors.lastname: Natsume
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan; PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kiyomitsu
authors.affiliation: Division of Mammalian Development, Genetic Strains Research Center, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan; Department of Genetics, SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Saga
authors.affiliation: Center of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, Japan; PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan; Department of Genetics, SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan. Electronic address: mkanemak@nig.ac.jp.
authors.forename: Masato T
authors.initials: MT
authors.lastname: Kanemaki
mesh_terms: D000818:Animals; D029681:Arabidopsis Proteins / Q000187:drug effects / Q000235:genetics*; D064113:CRISPR-Cas Systems; D053595:Embryonic Stem Cells / Q000378:metabolism; D044783:F-Box Proteins / Q000187:drug effects / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D020043:Genes, Essential; D045325:HCT116 Cells; D006801:Humans; D007210:Indoleacetic Acids / Q000494:pharmacology*; D051379:Mice; D059748:Proteolysis; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000235:genetics*; D017385:Sequence Homology
chemical_list: D029681:Arabidopsis Proteins; D044783:F-Box Proteins; D007210:Indoleacetic Acids; D011956:Receptors, Cell Surface; C501405:TIR1 protein, Arabidopsis