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

Toyoaki Natsume; Tomomi Kiyomitsu; Yumiko Saga; Masato T Kanemaki

doi:10.1016/j.celrep.2016.03.001

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

Cell Rep. 2016 Apr 5;15(1):210-218. doi: 10.1016/j.celrep.2016.03.001. Epub 2016 Mar 24.

Authors

Toyoaki Natsume¹, Tomomi Kiyomitsu², Yumiko Saga³, Masato T Kanemaki⁴

Affiliations

¹ Center of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
² 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.
³ 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.
⁴ 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.

PMID: 27052166
DOI: 10.1016/j.celrep.2016.03.001

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Arabidopsis Proteins / drug effects
Arabidopsis Proteins / genetics*
CRISPR-Cas Systems
Embryonic Stem Cells / metabolism
F-Box Proteins / drug effects
F-Box Proteins / genetics*
Gene Targeting / methods*
Genes, Essential*
HCT116 Cells
Humans
Indoleacetic Acids / pharmacology*
Mice
Proteolysis*
Receptors, Cell Surface / drug effects
Receptors, Cell Surface / genetics*
Sequence Homology

Substances

Arabidopsis Proteins
F-Box Proteins
Indoleacetic Acids
Receptors, Cell Surface
TIR1 protein, Arabidopsis