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

By Toyoaki Natsume, Tomomi Kiyomitsu, Y. Saga, Masato T. Kanemaki. Cell Reports, 2016

596 citations recorded by the source corpus.

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.

Subfields: Biotechnology, Molecular Biology, Oncology

View Paper: https://www.semanticscholar.org/paper/9f1d662ca0a8628b89ea750d435a276f3713e376

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Citations in the corpus

Cohesin loss eliminates all loop domains (2017, methodology): “In this system, constitutive expression of the auxinactivated ubiquitin ligase TIR1 leads, in the presence of auxin, to rapid degradation of proteins tagged with an AID domain.”
Targeted Protein Depletion Using the Auxin‐Inducible Degron 2 (AID2) System (2021, methodology): “Carry out standard western blotting using a 7.5% SDS-PAGE gel (see Kurien & Scofield, 2006; Current Protocols article: Ni, Xu, & Gallagher, 2016 for details).”
Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II, and Pol III (2020, result): “destabilization of endogenously tagged proteins is observed as reported previously [40].”