RRC ID 36024
Author Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT.
Title Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.
Journal Cell Rep
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.
Volume 15(1)
Pages 210-218
Published 2016-4-5
DOI 10.1016/j.celrep.2016.03.001
PII S2211-1247(16)30225-X
PMID 27052166
MeSH 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
IF 8.109
Times Cited 137
DNA material AAVS1 T2 CRIPR in pX330 (RDB13917) pMK232 (AAVS1 CMV-OsTIR1-PURO) (RDB13918) pMK243 (TetOne OsTIR1 AAVS1) (RDB13919) pMK277 (mClover-NeoR) (RDB13920) pMK278 (mClover-Hygro) (RDB13921) pMK279 (mClover-Bsr) (RDB13922) pMK280 (mCherry2-NeoR) (RDB13923) pMK281 (mCherry2-Hygro) (RDB13924) pMK282 (mCherry2-Bsr) (RDB13925) pMK283 (Stag-3FLAG-Neo) (RDB13926) pMK284 (Stag-FLAG-Hygro) (RDB13927) pMK285 (Stag-3FLAG-Bsr) (RDB13928) pMK286 (mAID-NeoR) (RDB13929) pMK287 (mAID-Hygro) (RDB13930) pMK288 (mAID-Bsr) (RDB13931) pMK289 (mAID-mClover-NeoR) (RDB13932) pMK290 (mAID-mClover-Hygro) (RDB13933) pMK291 (mAID-mClover-Bsr) (RDB13934) pMK292 (mAID-mCherry2-NeoR) (RDB13935) pMK293 (mAID-mCherry2-Hygro) (RDB13936) pMK294 (mAID-mCherry2-Bsr) (RDB13937)