To investigate the role of proteins in cells, gene knock down method is widely applied. Here Dr. Kanemaki offers a new method of reduction of interested proteins. He provides a novel method Auxin Inducible Degron (AID) System with which you can degrade proteins fused with a degron (a small domain that can induce protein degradation) in a very short period of time.
DNA Resource
The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.
Yesbolatova, A., Saito, Y., Kitamoto, N., Makino-Itou, H., Ajima, R., Nakano, R., Nakaoka, H., Fukui, K., Gamo, K., Tominari, Y., Takeuchi, H., Saga, Y., Hayashi, K., Kanemaki, M.T.
Nat. Commun. 11: 5701, 2020.
Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole.
Yesbolatova, A., Natsume, T., Hayashi, K.I., Kanemaki, M.T.
Methods pii: S1046-2023 (18) 30331-1, 2019. PMID: 31026591.
Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.
Natsume, T., Kiyomitsu, T., Saga, Y., Kanemaki, M.T.
Cell Reports 15: 210-218, 2016. PMID: 27052166.
Table 1. CsTIR1 and AtAFB2 expression vectors and helper plasmid
a. CsTIR1 and AtAFB2 expression vectors
Catalog# | Name of clone | Short description |
---|---|---|
RDB13918 | pMK232 | OsTIR1 expression vector, PURO |
RDB16811 | pMK364 | OsTIR1 expression vector, loxP-PURO-loxP |
RDB18337 | pMK381![]() |
OsTIR1 F74G expression vector, loxP-PURO-loxP |
RDB18347 | pMK233![]() |
AtAFB2 expression vector, loxP-PURO-loxP |
b. Helper plasmid for knock-in a expression vector to the human AAVS1 site
RDB13917 | AAVS1 T2 CRIPR in pX330 | A CRISPR/Cas plasmid for making DNA dounle-strand break at the human AAVS1 locus. |
c. CsTIR1 expression vector of yeast
RDB18341 | pMK198![]() |
GAL-OsTIR1 expression vector of yeast |
RDB18342 | pMK419![]() |
GAL-OsTIR1(F74G) expression vector of yeast |
RDB18343 | pMK425![]() |
GAL-OsTIR1(F74A) expression vector of yeast |
Table 2. Tags
a. mAID tags, N-terminal tagging
Selection marker |
Attached tag at the N-terminus | |||
---|---|---|---|---|
mAID | mAID-mClover | mAID-mCherry2 | mAID-Nluc | |
Hygro | pMK344 | pMK345 | pMK346 | pMK401 |
BSD | pMK347 | pMK348 | pMK349 | pMK407 |
b. mAID tags, C-terminal tagging
Selection marker |
Attached tag at the C-terminus | |||
---|---|---|---|---|
mAID | mAID-mClover | mAID-mCherry2 | mAID-Nluc | |
Neo | pMK286 | pMK289 | pMK292 | pMK394 |
Hygro | pMK287 | pMK290 | pMK293 | pMK408 |
BSD | pMK391 | pMK392 | pMK393 | pMK409 |
Bsr | pMK288 | pMK291 | pMK294 |
Table 3. Tags
a. Tags without mAID, N-terminal tagging
Selection marker |
Attached tag at the N-terminus | ||
---|---|---|---|
mClover | mCherry2 | S tag-3FLAG | |
Hygro | pMK376 | pMK377 | pMK375 |
b. Tags without mAID, C-terminal tagging
Selection marker |
Attached tag at the C-terminus | ||
---|---|---|---|
mClover | mCherry2 | S tag-3FLAG | |
Neo | pMK277 | pMK280 | pMK283 |
Hygro | pMK278 | pMK281 | pMK284 |
BSD | pMK388 | pMK389 | pMK390 |
Bsr | pMK279 | pMK282 | pMK285 |
Table 4.
mAID and mIAA7 tagging specific gene
Gene | Helper | Tagging with Hygro marker | Tagging with Neo marker |
---|---|---|---|
BRD4 | BRD4-N CRISPR | mAID-BRD4 donor | |
CTCF | CTCF-C CRISPR | CTCF-mAC donor (Hygro) | CTCF-mAC donor (Neo) |
DHC1 | DHC1 CRISPR | pMK297 | pMK296 |
DHC1 | DHC1 CRISPR | DHC1-mIAA7 Hygro | DHC1-mIAA7 Neo |
RAD21 | RAD21 C-tag CIRPSR | pMK265 | pMK262 |
SMC2 | SMC2 CRISPR | SMC2-mAC donor (Hygro) | |
TOP2A | pMK312 | pMK322 | pMK321 |
Table 5.
Simultaneous expression of OsTIR1 and mAID-EGFP
Catalog no. | Name of clone | Short description |
---|---|---|
RDB18338 | pAAV-hSyn-OsTIR1(WT) | Adeno-associated virus (AAV) expression vector of OsTIR1(wild type) and mAID-EGFP-NES marker with hSyn promoter. |
RDB18339 | pAAV-hSyn-OsTIR1(F74G) | Adeno-associated virus (AAV) expression vector of OsTIR1(F74G) and mAID-EGFP-NES marker with hSyn promoter. |
RDB18340 | pAAV-hSyn-OsTIR1(F74A) | Adeno-associated virus (AAV) expression vector of OsTIR1(F74A) and mAID-EGFP-NES marker with hSyn promoter. |
RDB18333 | pAY7 | TOL2 expression vector of OsTIR1(WT) and mAID-EGFP-NLS marker. |
RDB18334 | pAY15 | TOL2 expression vector of OsTIR1(F74G) and mAID-EGFP-NLS marker. |
RDB18335 | pAY19 | TOL2 expression vector of OsTIR1(F74A) and mAID-EGFP-NLS marker. |
RDB18363 | pMK427 | TOL2 expression vector of OsTIR1(WT) and mAID-EGFP-Nluc marker. |
RDB18364 | pMK411 | TOL2 expression vector of OsTIR1(F74G) and mAID-EGFP-Nluc marker. |
An auxin-based degron system for the rapid depletion of proteins in nonplant cells.
Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., Kanemaki, M.
Nat. Methods., 6, 917-922, 2009. PMID: 19915560.
Catalog# | Name of clone | Running title | Type |
---|---|---|---|
RDB08468 | pNHK60 | Expression clone for an auxin-based degron (AID) system | Plasmid |
RDB08469 | pMK106 | Expression clone for an auxin-based degron (AID) system | Plasmid |
RDB08470 | pMK107 | Expression clone for an auxin-based degron (AID) system | Plasmid |
Please refer
Supplementary Figure 11 for pMK106 (aid-CENPH)
Supplementary Figure 11 for pMK107 (CENPH-aid)
Supplementary figure 10 for pNHK60 (pIRES OsTIR1-EGFP-IAA17)

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References & Related Articles
- Yesbolatova, A., Natsume, T., Hayashi, K.I., Kanemaki, M.T. Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole. Methods pii: S1046-2023 (18) 30331-1, 2019. PMID: 31026591.
- Research highlights
Development of a CRISPR/Cas9-based tagging method and a degradation inhibitor for the auxin-inducible degron (AID) system - Natsume, T., Nishimura, K., Minocherhomji, S., Bhowmick, R., Hickson, I.D., Kanemaki, M.T. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis. Genes Dev. 31 (8): 816-829, 2017.. PMID: 28487407.
- Natsume, T., Kiyomitsu, T., Saga, Y., Kanemaki, M.T. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell Reports 15: 210-218, 2016. PMID: 27052166.
- Takagi, M., Natsume, T., Kanemaki, M.T., Imamoto, N. Perichromosomal protein Ki67 supports mitotic chromosome architecture. Genes Cells 21 (10): 1113-1124, 2016. PMID: 27610954.
- Samejima, K., Ogawa, H., Ageichik, A.V., Peterson, K.L., Kaufmann, S.H., Kanemaki, M.T., Earnshaw, W.C. Auxin-induced Rapid Degradation of Inhibitor of Caspase-activated DNase (ICAD) Induces Apoptotic DNA Fragmentation, Caspase Activation, and Cell Death: A CELL SUICIDE MODULE. J. Biol. Chem. 289 (45): 31617-31623, 2014. PMID: 25248749
- Rodriguez-Bravo, V., Maciejowski, J., Corona, J., Buch, H.K., Collin, P., Kanemaki, M.T., Shah, J.V., Jallepalli, P.V. Nuclear pores protect genome integrity by assembling a premitotic and Mad1-dependent anaphase inhibitor. Cell 156 (5): 1017-1031, 2014. PMID: 24581499
- Kanemaki, M.T. Frontiers of protein expression control with conditional degrons. Eur. J. Physiol., 465, 419-425, 2013. PMID: 23271452.
- Holland, A.J., Fachinetti, D., Han, J.S., Cleveland, D.W. Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 109 (49): E3350-E3357, 2012. PMID: 23150568
- Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods., 6, 917-922, 2009. PMID: 19915560.
- Matato Kanemaki Lab Web Page
- BioResource Now! Issue 7, July 2011 (PDF, in Japanese)
- BioResource Now! Issue 7, July 2011 (PDF, in English)
(GRP0016e 2012.11.07 T.M.)
2020.12.22