The ability to deplete a protein of interest is critical for dissecting cellular processes. Traditional methods of protein depletion are often slow acting, which can be problematic when characterizing a cellular process that occurs within a short period of time, such as mitosis. Furthermore, these methods are usually not reversible. Recent advances to achieve protein depletion function by inducibly trafficking proteins of interest to an endogenous E3 ubiquitin ligase complex to promote ubiquitination and subsequent degradation by the proteasome. One of these systems, the auxin-inducible degron (AID) system, has been shown to permit rapid and inducible degradation of AID-tagged target proteins in mammalian cells. The AID system can control the abundance of a diverse set of cellular proteins, including those contained within protein complexes, and is active in all phases of the cell cycle. Here we discuss considerations for the successful implementation of the AID system and describe a protocol using CRISPR/Cas9 to achieve biallelic insertion of an AID in human cells. This method can also be adapted to insert other tags, such as fluorescent proteins, at defined genomic locations.
Keywords: Auxin; Degradation; IAA; Mitosis; Proteasome.
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