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

• Short-arm donors are used for tagging in human HCT116 and mouse ES cells
• Conditional AID mutants are generated in HCT116 cell lines expressing OsTIR1
• Essential proteins fused with AID are depleted rapidly upon the addition of auxin
• The developed method is simple and scalable for construction of AID mutants

SummaryStudying 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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