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

Abstract

Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional, universal, and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use nanobodies and single chain variable fragments (scFv), which recognize small epitopes, enabling fluorescent visualization and selective degradation of protein targets in vivo. Furthermore, we delineate a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad multifunctionality. We use GEARs to examine the native behaviour 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.

Novel cell states arise in embryonic cells devoid of key reprogramming factors

“…A truncated human CD4 sequence (corresponding to the sequence identified by hCD4 MicroBeads, Miltenyi Biotec) was cloned into a pCS2+ vector containing an SP6 promoter, a stabilizing 3’UTR derived from the zebrafish prrg2 endogenous gene (Boswell et al ., 2023), and an SV40 pA.…”

“…From single cell RNA sequencing experiments, we noted an over-representation of cell types of ectodermal origin in WT donor cells relative to WT host cell populations, although all tissue types were represented, indicating a potential technical bias introduced at the site of transplantation (as shown in Figures 2E and 4A) (Kimmel, Warga and Schilling, 1990). hCD4 magnetic microbeads-mediated cell enrichment of transplanted cells A truncated human CD4 sequence (corresponding to the sequence identified by hCD4 MicroBeads, Miltenyi Biotec) was cloned into a pCS2+ vector containing an SP6 promoter, a stabilizing 3'UTR derived from the zebrafish prrg2 endogenous gene (Boswell et al, 2023), and an SV40 pA. The truncated hCD4 sequence was…”