Genetically encoded affinity reagents (GEARs): A toolkit for visualizing and manipulating endogenous protein function in vivo

Probing endogenous protein localization and function in vivo remains challengingdue to laborious gene targeting and monofunctional alleles. Here, we develop amultifunctional and adaptable toolkit based on genetically encoded affinityreagents (GEARs). GEARs use small epitopes recognized by nanobodies and singlechain variable fragments to enable fluorescent visualization, manipulation anddegradation of protein targets in vivo. Furthermore, we outline aCRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility forproducing knock-in alleles that have broad applications. We use GEARs toexamine the native behaviour of the pioneer transcription factor Nanog and theplanar cell polarity protein Vangl2 during early zebrafish development. Together,this toolkit provides a versatile system for probing and perturbing endogenousprotein function while circumventing challenges associated with conventionalgene targeting and is broadly available to the model organism community.