Genetically Encoded Affinity Reagents Are a Toolkit for Visualizing and Manipulating Endogenous Protein Function In Vivo

• The reagents and datasets provided below were generated by Boswell et al. (2025). Please cite the manuscript if you utilize any of these resources.
• Stable knock-in and transgenic lines generated in this study are maintained at Yale University and available upon request.
• For questions please email antonio.giraldez@yale.edu or curtis.boswell@yale.edu.

Protocols

• Epitope tag integration for zebrafish

GEARs Constructs

The following genetically encoded affinity reagent (GEAR) constructs are available upon request. For zebrafish experiments, we recommend using the ALFA epitope and its associated reagents.

Target Epitope	Fusion	Construct	Description	Plasmid Map
HA	EGFP	pCS2+ EGFP-FbHA boomUTR	EGFP fused to HA frankenbody (15F11) with stabilizing UTR	map
	mNeonGreen	pCS2+ mNG-FbHA boomUTR	mNeonGreen fused to HA frankenbody (15F11) with stabilizing UTR	map
	mScarlet-I	pCS2+ mScarleti-FbHA boomUTR	mScarlet-i fused to HA frankenbody (15F11) with stabilizing UTR	map
	mTagBFP2	pCS2+ mTagBFP2-FbHA boomUTR	mTagBFP2 fused to HA frankenbody (15F11) with stabilizing UTR	map
	HaloTag	pCS2+ Halo-FbHA boomUTR	HaloTag fused to HA frankenbody (15F11) with stabilizing UTR	map
	fbxw11b	pCS2+ HAgrad	Zebrafish fbxw11b fused to HA frankenbody (15F11) with stabilizing UTR	map
FLAG	EGFP	pCS2+ EGFP-FbFLAG boomUTR	EGFP fused to FLAG frankenbody with stabilizing UTR	map
	mNeonGreen	pCS2+ mNG-FbFLAG boomUTR	mNeonGreen fused to FLAG frankenbody with stabilizing UTR	map
	mScarlet-I	pCS2+ mScarleti-FbFLAG boomUTR	mScarlet-i fused to FLAG frankenbody with stabilizing UTR	map
	mTagBFP2	pCS2+ mTagBFP2-FbFLAG boomUTR	mTagBFP2 fused to FLAG frankenbody with stabilizing UTR	map
	HaloTag	pCS2+ Halo-FbFLAG boomUTR	HaloTag fused to FLAG frankenbody with stabilizing UTR	map
	fbxw11b	pCS2+ FLAGgrad	Zebrafish fbxw11b fused to FLAG frankenbody with stabilizing UTR	map
ALFA	EGFP	pCS2+ EGFP-NbALFA boomUTR	EGFP fused to ALFA nanobody with stabilizing UTR	map
	mNeonGreen	pCS2+ mNG-NbALFA boomUTR	mNeonGreen fused to ALFA nanobody with stabilizing UTR	map
	mScarlet-I	pCS2+ mScarleti-NbALFA boomUTR	mScarlet-i fused to ALFA nanobody with stabilizing UTR	map
	mScarlet-I3	pCS2+ mScarleti3-NbALFA boomUTR	mScarlet-i3 fused to ALFA nanobody with stabilizing UTR	map
	mTagBFP2	pCS2+ mTagBFP2-NbALFA boomUTR	mTagBFP2 fused to ALFA nanobody with stabilizing UTR	map
	HaloTag	pCS2+ Halo-NbALFA boomUTR	HaloTag fused to ALFA nanobody with stabilizing UTR	map
	fbxw11b	pCS2+ ALFAgrad	Zebrafish fbxw11b fused to ALFA nanobody with stabilizing UTR	map
VHH05	EGFP	pCS2+ EGFP-NbVHH05 boomUTR	EGFP fused to VHH05 nanobody with stabilizing UTR	map
	mNeonGreen	pCS2+ mNG-NbVHH05 boomUTR	mNeonGreen fused to VHH05 nanobody with stabilizing UTR	map
	mScarlet-I	pCS2+ mScarleti-NbVHH05 boomUTR	mScarlet-i fused to VHH05 nanobody with stabilizing UTR	map
	mTagBFP2	pCS2+ mTagBFP2-NbVHH05 boomUTR	mTagBFP2 fused to VHH05 nanobody with stabilizing UTR	map
	HaloTag	pCS2+ Halo-NbVHH05 boomUTR	HaloTag fused to VHH05 nanobody with stabilizing UTR	map
	fbxw11b	pCS2+ VHH05grad	Zebrafish fbxw11b fused to VHH05 nanobody with stabilizing UTR	map
127d01	EGFP	pCS2+ EGFP-Nb127d01 boomUTR	EGFP fused to 127D01 nanobody with stabilizing UTR	map
	mNeonGreen	pCS2+ mNG-Nb127d01 boomUTR	mNeonGreen fused to 127D01 nanobody with stabilizing UTR	map
	mScarlet-I	pCS2+ mScarleti-Nb127d01 boomUTR	mScarlet-i fused to 127D01 nanobody with stabilizing UTR	map
	mTagBFP2	pCS2+ mTagBFP2-Nb127d01 boomUTR	mTagBFP2 fused to 127D01 nanobody with stabilizing UTR	map
	HaloTag	pCS2+ Halo-Nb127d01 boomUTR	HaloTag fused to 127D01 nanobody with stabilizing UTR	map
	fbxw11b	pCS2+ 127d01grad	Zebrafish fbxw11b fused to 127D01 nanobody with stabilizing UTR	map
Sun	EGFP	pCS2+ EGFP-FbSun boomUTR	EGFP fused to Sun (GCN4) scFv with stabilizing UTR	map
	fbxw11b	pCS2+ Sungrad	Zebrafish fbxw11b fused to Sun (GCN4) scFv with stabilizing UTR	map
Moon	EGFP	pCS2+ EGFP-NbMoon boomUTR	EGFP fused to Moon (gp41) nanobody with stabilizing UTR	map
	fbxw11b	pCS2+ Moongrad	Zebrafish fbxw11b fused to Moon (gp41) nanobody with stabilizing UTR	map

Unpublished GEARs Constructs

The following genetically encoded affinity reagent (GEAR) constructs are available upon request.

Target Epitope	Fusion	Construct	Description	Plasmid Map

Knock-In Lines

Knock-in alleles were generated via CRISPR/Cas9-mediated epitope tag integration. All homozygous knock-in embryos develop normally and are phenotypically indistinguishable from wildtype at 24 hpf:

Gene	Epitope Tag	Yale Allele	Allele
nanog	ALFA	ya3329	ALFA-nanog
nanog	VHH05	ya3330	VHH05-nanog
nanog	127d01	ya3331	127d01-nanog
vangl2	ALFA	ya3332	ALFA-vangl2
vangl2	VHH05	ya3333	VHH05-vangl2
vangl2	127d01	ya3334	127d01-vangl2
pou5f3	ALFA	ya3335	ALFA-pou5f3
dicer	ALFA	ya3336	ALFA-dicer

Transgenic Zebrafish Lines

Line	Yale Allele	Description
Tg(actb2:EGFP-NbALFA)	ya3326	Ubiquitous expression of EGFP-tagged anti-ALFA nanobody
Tg(hsp70l:ALFAgrad,myl7:EGFP)	ya3327	Heat-shock inducible ALFA-tag degrader
Tg(fli1a:ALFAgrad-P2A mCherry)	ya3328	Endothelial-specific ALFA-tag degrader with mCherry reporter

Unpublished Transgenic Zebrafish Lines

Line	Yale Allele	Description

Reagent Design Resources

Tool	Purpose	Link
CRISPRscan	sgRNA design for CRISPR/Cas9 knock-in	crisprscan.org
ICE Analysis (Synthego)	Cleavage efficiency estimation	ice.synthego.com
iCodon	Codon optimization for zebrafish expression	icodon.com