A chemically inducible, leakless Cre recombinase by split-protein-based efficient and enhanced degradation (SPEED)
Year: 2025
Type: article
Abstract: Chemically inducible DNA recombination systems are a very attractive tool for implementing potent genetic applications. However, conventional systems still suffer from leaky properties in the absence of chemicals. Here we describe a chemically inducible, leakless destabilized Cre recombinase (SPEED-Cre) based on a new approach, split-protein-based efficient and enhanced degradation (SPEED), that consists of a self-assembling split-Cre tagged with a destabilizing domain (DD) mutant from the Escherichia coli dihydrofolate reductase that is stabilized by the antibiotic ligand trimethoprim (TMP). We demonstrate that SPEED-Cre has no significant leak activity of background DNA recombination in the absence of TMP; nevertheless, it enables full induction of TMP-dependent Cre-loxP recombination in human cells and living mice. We also demonstrate the general applicability of the SPEED approach, which can be widely applied to other proteins, by showing high TMP-dependent recombination performances of destabilized Flp, VCre and Dre recombinases based on the SPEED approach. This robust platform technology will greatly enhance chemogenetic applications for genome engineering in living systems. A combination of self-assembling split-Cre with a destabilizing domain system is found to be an effective way to improve the degradation efficiency of destabilized Cre in the absence of Trimethoprim (Tmp) while maintaining efficient TMP-inducible DNA recombination.
Source: Communications Biology
Authors: Dewen Cai, Fuun Kawano, Takahiro Otabe, Koji Hashimoto, Moritoshi Sato
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $4,190
Funders: Naito Foundation, Takeda Science Foundation, Japan Agency for Medical Research and Development, Ministry of Education, Culture, Sports, Science and Technology, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan Society for the Promotion of Science, Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Precursory Research for Embryonic Science and Technology
Awards: G1605823172, 反応漏れの無い化合物誘導型遺伝子組換え技術の開発, G2521286739, G2576085165, G2911495042, G3493588279, Development and applications of optogenetic tools for controlling transcription of short-chain RNA in mammals, G4073961370, Development of a red-light controllable gene expression system in combination with signal amplification approaches, Development of cellular tools based optogenetic technology, G4276273683, G5070855035, 光操作に基づくin vivo遺伝子治療技術の創出, G59247851, G6718509927, G6884320423, G7415814815, Development and application of optogenetic cell labeling technology
OpenAlex ID: W4417303228