Engineering Tools for Rapid Loss of Protein Function with Spatio-Temporal Control in Zebrafish

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PROJECT SUMMARY In zebrafish, the genetic toolkit is limited to global and tissue-specific gene inactivation. However, often it is desirable to inactivate genes in specific tissues and at specific times. Moreover, phenotypes do not become evident until pre-existing protein product from the targeted gene has decayed. This lag can be many hours or even days. This is a problem when studying rapid biological processes or genes that are required for cell survival. In recent years, several strategies have been developed to overcome these limitations. Most of the strategies target the protein gene product directly, typically by tagging the protein with a degron that can be induced to degrade the tagged protein. We recently adapted the deGradFP system from flies to zebrafish and called it zGrad. zGrad targets GFP-tagged proteins for degradation and reveals loss-of-function phenotypes. zGrad consists of a fusion of an anti-GFP nanobody to a F-box domain. zGrad binds GFP-tagged proteins through its anti-GFP nanobody part and targets the tagged protein to the E3 ubiquitin ligase complex for degradation through its F-box domain. In its current form, zGrad can be used to degrade proteins with either spatial or temporal control, but not both. Here we propose to engineer significant improvements to the zGrad system. Specifically, we will (1) expand zGrad so that it can be used to degrade proteins with combined spatial and temporal control, and (2) engineer zGrad so that it is regulated through light to induce degradation in specific cells and subcellular compartments at desired times. These improvements will make zGrad an extremely powerful and versatile system for inactivating genes rapidly in specific cells at specific times in zebrafish, and would provide proof-of-principle that the deGradFP/zGrad method could be adapted to function in any system.