A robust dual gene ON–OFF toggle directed by two independent promoter–degron pairs
| Autor: | Tsz Kwan Yeung, Sehong Kim, Hoi Tang Ma, Randy Y. C. Poon |
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| Rok vydání: | 2023 |
| Předmět: | |
| Zdroj: | Journal of Cell Science. 136 |
| ISSN: | 1477-9137 0021-9533 |
| Popis: | Switching genes on and off on cue is a cornerstone for understanding gene functions. One contemporary approach for loss-of-function studies of essential genes involves CRISPR-mediated knockout of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect in mammalian cell lines. A broadening of this approach would involve simultaneously switching on a second construct to interrogate the functions of a gene in the pathway. In this study, we developed a pair of switches that were independently controlled by both inducible promoters and degrons, enabling the toggling between two constructs with comparable kinetics and tightness. The gene-OFF switch was based on TRE transcriptional control coupled with auxin-induced degron-mediated proteolysis. A second independently controlled gene-ON switch was based on a modified ecdysone promoter and mutated FKBP12-derived destabilization domain degron, allowing acute and tuneable gene activation. This platform facilitates efficient generation of knockout cell lines containing a two-gene switch that is regulated tightly and can be flipped within a fraction of the time of a cell cycle. |
| Databáze: | OpenAIRE |
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