Kanemaki Group • Molecular Cell Engineering Laboratory

A new genetics of human cells for the study of DNA replication


Research Summary

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–Cas9-based genome-editing technology with the AID system, we can now make human conditional mutants. We continue to make new technologies related to AID and, by applying them, 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 CRISPR-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.

Selected Publications

Yesbolatova A, Natsume T, Hayashi KI, Kanemaki MT. Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole. Methods. 2019 Jul 15;164-165:73-80.

Natsume T, Nishimura K, Minocherhomji S, Bhowmick R, Hickson ID, Kanemaki MT. Acute inactivation of the replicative helicase in human cells triggers MCM8- 9-dependent DNA synthesis. Genes Dev. 2017 Apr 15;31(8):816-829.

Natsume T, Kanemaki MT. Conditional Degrons for Controlling Protein Expression at the Protein Level. Annu Rev Genet. 2017 Nov 27;51:83-102.

Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell Rep. 2016 Apr 5;15(1):210-218.

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