Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors | Zendy

Toyoaki Natsume | Zendy; Tomomi Kiyomitsu | Zendy; Yumiko Saga | Zendy; Masato T. Kanemaki | Zendy

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Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

Author(s) -

Toyoaki Natsume,

Tomomi Kiyomitsu,

Yumiko Saga,

Masato T. Kanemaki

Publication year - 2016

Publication title -

cell reports

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 6.264

H-Index - 154

eISSN - 2639-1856

pISSN - 2211-1247

DOI - 10.1016/j.celrep.2016.03.001

Subject(s) - degron , crispr , biology , microbiology and biotechnology , phenotype , endogeny , embryonic stem cell , cytoplasm , nuclear protein , function (biology) , genetics , biochemistry , gene , transcription factor , ubiquitin ligase , ubiquitin

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.

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