Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo

Curtis W. Boswell (), Caroline Hoppe, Alice Sherrard, Liyun Miao, Mina L. Kojima, Pieter Martino, Ning Zhao, Timothy J. Stasevich, Stefania Nicoli and Antonio J. Giraldez ()
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Curtis W. Boswell: Yale University School of Medicine
Caroline Hoppe: Yale University School of Medicine
Alice Sherrard: Yale University School of Medicine
Liyun Miao: Yale University School of Medicine
Mina L. Kojima: Yale University School of Medicine
Pieter Martino: Yale University School of Medicine
Ning Zhao: University of Colorado-Anschutz Medical Campus
Timothy J. Stasevich: Colorado State University
Stefania Nicoli: Yale University School of Medicine
Antonio J. Giraldez: Yale University School of Medicine

Nature Communications, 2025, vol. 16, issue 1, 1-20

Abstract: Abstract Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use small epitopes recognized by nanobodies and single chain variable fragments to enable fluorescent visualization, manipulation and degradation of protein targets in vivo. Furthermore, we outline a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad applications. We use GEARs to examine the native behavior of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.

Date: 2025
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DOI: 10.1038/s41467-025-61003-w

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