A single-chain antibody-based AID2 system for conditional degradation of GFP-tagged and untagged proteins

Islam, M.; Negishi, T.; Kitamoto, N.; Hatoyama, Y.; Gamo, K.; Hayashi, K.-i.; Kanemaki, M. T.

doi:10.1101/2025.01.21.633519

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bioRxiv · 10.1101/2025.01.21.633519

A single-chain antibody-based AID2 system for conditional degradation of GFP-tagged and untagged proteins

Islam, M.·Negishi, T.·Kitamoto, N.·Hatoyama, Y.·Gamo, K.·Hayashi, K.-i.·Kanemaki, M. T.

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Abstract

Protein knockdown using an improved auxin-inducible degron (AID2) technology has proven to be a powerful tool for studying protein function. The current approach requires the fusion of target proteins with a degron tag, a process typically achieved through CRISPR knock-in. However, knock-in remains challenging in non-model organisms and humans, limiting the broader applicability of AID2. To overcome this limitation, we developed a single-chain antibody AID2 (scAb-AID2) system. This approach employs an adaptor composed of a single-chain antibody fused with a degron, which recognises a target protein and induces rapid degradation in the presence of the inducer 5-Ph-IAA. We demonstrated that scAb-AID2, in combination with an anti-GFP nanobody, degraded GFP-fused proteins in human cells and C. elegans. Furthermore, we showed that endogenous p53 and H/K-RAS were conditionally degraded in cells expressing an adaptor encoding an anti-p53 nanobody and -RAS monobody, respectively, and led to aphidicolin sensitivity in cell culture and growth inhibition in mouse xenografts. This study paves the way for broader application of AID2-based target depletion in model and non-model organisms and for advancing therapeutic strategies.

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Islam, M., Negishi, T., Kitamoto, N., Hatoyama, Y., Gamo, K., Hayashi, K.-i., Kanemaki, M. T.. 2025-01-24. A single-chain antibody-based AID2 system for conditional degradation of GFP-tagged and untagged proteins. https://doi.org/10.1101/2025.01.21.633519

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