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Samal Tazhibayeva (D4, Department of Genetics, SOKENDAI) in Multicellular Organization Laboratory received the Poster Award (Honorable Mention） at the 25^th International Worm Meeting held in UC Davis USA on June 28^th-July 2^nd 2025. She also received the grand prize in Art Show during the meeting by her painting.

・ Awarded presentation title: CWN-2/Wnt localization on seam cells challenges gradient-dependent polarity regulation by Wnt

・ 25th International Worm Meeting

・ Sawa Group • Multicellular Organization Laboratory

Kanemaki Group / Molecular Cell Engineering Laboratory
Sawa Group / Multicellular Organization Laboratory

A Single-Chain Antibody-Based AID2 System for Conditional Degradation of GFP-Tagged and Untagged Proteins

Moutushi Islam, Takefumi Negishi, Naomi Kitamoto, Yuki Hatoyama, Kanae Gamo, Ken-ichiro Hayashi, and Masato T. Kanemaki*
*corresponding author

Journal of Cell Science (2025)  jcs.263961 DOI:10.1242/jcs.263961

By inducing rapid degradation of a target protein in living cells, it becomes possible to observe the effects caused by the loss of that protein’s function. The auxin-inducible degron (AID) technology developed by the Kanemaki Laboratory has been widely used for functional studies of various proteins. The latest improved version, AID2, allows precise and efficient control of target protein degradation. However, AID2 requires genome editing to fuse the degron tag to the target protein, which has limited its application to non-model organisms and clinical settings. To overcome this limitation, the Kanemaki Laboratory successfully developed the single-chain antibody AID2 (scAb-AID2) system, which enables degradation without the need to tag the target protein by utilizing single-chain antibodies.

As a proof-of-concept experiment, they employed a GFP nanobody, a single-chain antibody that recognizes GFP. An adapter consisting of the GFP nanobody fused to a degron tag was introduced along with the ubiquitin ligase OsTIR1(F74G) into human cultured cells and C. elegans expressing various GFP-tagged proteins. Upon addition of the degradation-inducing ligand 5-Ph-IAA, these GFP-fusion proteins were successfully degraded.

Furthermore, they designed adapters fused with degron tags using single-chain antibodies targeting the tumor suppressor p53 and the growth-promoting factors H/K-RAS. These adapters and OsTIR1(F74G) were introduced into human cultured cells. Treatment of these cells with 5-Ph-IAA induced degradation of p53 and H/K-RAS. Notably, degradation of p53 increased the cells’ sensitivity to a replication stress-inducing agent, while tumors formed from cells in which H/K-RAS was degraded showed suppressed tumor formation.

The scAb-AID2 system overcomes the limitation of degron tagging required in the conventional AID2 system and opens new avenues for both basic life science research and clinical applications through targeted protein degradation.

In connection with this paper, the first author, SOKENDAI student Moutushi Islam, was featured in the First Person section of the Journal of Cell Science. In addition, the paper was selected as a Research Highlight in the same journal.

This work was supported by JSPS KAKENHI grant (JP23K05836) to Takefumi Negishi, and JSPS KAKENHI grants (JP21H04719, JP23H04925 and JP25H00979) and JST CREST grant (JPMJCR21E6) to Masato T. Kanemaki.

Figure: Single-chain antibody AID2 system enables degradation of untagged proteins in C. elegans, mammalian cells and mouse in a 5-Ph-IAA dependent manner.

• First person
• Antibodies AID protein degradation

■ Technical term

• Auxin-Inducible Degron (AID) Technology:
  A protein degradation technology developed by the Kanemaki Laboratory. It was created by transplanting the auxin-dependent degradation system, originally found in plants, into non-plant cells. The improved version, AID2, has been applied to various cells and organisms, including yeast, cultured cells, C. elegans, and mice.
• Single-chain Antibody (scAb):
  Unlike conventional antibodies composed of multiple subunits, a single-chain antibody consists of a single polypeptide chain. A well-known example is the nanobody derived from a variable region of the antibody heavy chain produced by the camelid species. In addition, various artificial single-chain antibodies has been developed.