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.


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


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