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bioRxiv · 10.1101/2023.03.26.534268

One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging

Li, S.·Wang, Y.·Stoel, M. v. d.·Zhou, X.·Madhusudan, S.·Kanerva, K.·Nguyen, V. D.·Eskici, N.·Olkkonen, V. M.·Zhou, Y.·Raivio, T.·Ikonen, E.
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Abstract

Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

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Li, S., Wang, Y., Stoel, M. v. d., Zhou, X., Madhusudan, S., Kanerva, K., Nguyen, V. D., Eskici, N., Olkkonen, V. M., Zhou, Y., Raivio, T., Ikonen, E.. 2023-03-27. One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging. https://doi.org/10.1101/2023.03.26.534268

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