We established the auxin-inducible degron (AID) technology, by which the expression of a protein of interest can be rapidly controlled by the addition of a plant hormone, auxin. By combining the CRISPR–Cas-based genome-editing technology with the AID system, we can now make human conditional mutants, in which a protein of interest can be depleted in a half-life of less than 30 min. By applying the AID technology, we wish to understand how genomic DNA is maintained in human cells. In particular, we are focusing on the relationship between DNA replication and other DNA transactions.
The auxin-inducible degron (AID) technology
(A) The AID tag was inserted to a target gene by CRIPSPR-Cas9 mediated knock-in using a donor plasmid. In the presence of auxin, the fusion protein will be recognized by an ubiquitin ligase with A containing plant-derived TIR1 for rapid degradation.
(B) RAD21, a component of cohesion, was fused with AID and fluorescent tags. RAD21 is depleted rapidly upon auxin addition.
(C) Quantification of the level of RAD21.
Natsume, T., Nishimura, K., Minocherhomji, S., Bhowmick, R., Hickson, I. D., and Kanemaki, M. T. (2017). Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis. Genes Dev 31, 816-829.
Natsume, T., and Kanemaki, M. T. (2017). Conditional degrons for controlling protein expression at the protein level. Annu Rev Genet 51, 83-102.
Natsume, T., Kiyomitsu, T., Saga, Y., and Kanemaki, M.T. (2016). Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Reports 15, 210-218.