Dataset Information

HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells.

ABSTRACT: Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.

SUBMITTER: Li S

PROVIDER: S-EPMC10895734 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells.

Li Shiqian S Wang Yafei Y van der Stoel Miesje M Zhou Xin X Madhusudan Shrinidhi S Kanerva Kristiina K Nguyen Van Dien VD Eskici Nazli N Olkkonen Vesa M VM Zhou You Y Raivio Taneli T Ikonen Elina E

Genome biology 20240226 1

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its ap ...[more]

PMID: 38409044

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Project description:Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.

Dataset Information

HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells.

Publications

HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells.

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