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 it’s associated reagents.

Target Epitope	Fusion	Construct	Description	Plasmid Map
HA	EGFP	pCS2+ EGFP-FbHA boomUTR		map
	mNeonGreen	pCS2+ mNG-FbHA boomUTR		map
	mScarlet-I	pCS2+ mScarleti-FbHA boomUTR		map
	mTagBFP2	pCS2+ mTagBFP2-FbHA boomUTR		map
	HaloTag	pCS2+ Halo-FbHA boomUTR		map
	fbxw11b	pCS2+ HAgrad		map
FLAG	EGFP	pCS2+ EGFP-FbFLAG boomUTR		map
	mNeonGreen	pCS2+ mNG-FbFLAG boomUTR		map
	mScarlet-I	pCS2+ mScarleti-FbFLAG boomUTR		map
	mTagBFP2	pCS2+ mTagBFP2-FbFLAG boomUTR		map
	HaloTag	pCS2+ Halo-FbFLAG boomUTR		map
	fbxw11b	pCS2+ FLAGgrad		map
ALFA	EGFP	pCS2+ EGFP-NbALFA boomUTR		map
	mNeonGreen	pCS2+ mNG-NbALFA boomUTR		map
	mScarlet-I	pCS2+ mScarleti-NbALFA boomUTR		map
	mScarlet-I3	pCS2+ mScarleti3-NbALFA boomUTR		map
	mTagBFP2	pCS2+ mTagBFP2-NbALFA boomUTR		map
	HaloTag	pCS2+ Halo-NbALFA boomUTR		map
	fbxw11b	pCS2+ ALFAgrad		map
VHH05	EGFP	pCS2+ EGFP-NbVHH05 boomUTR		map
	mNeonGreen	pCS2+ mNG-NbVHH05 boomUTR		map
	mScarlet-I	pCS2+ mScarleti-NbVHH05 boomUTR		map
	mTagBFP2	pCS2+ mTagBFP2-NbVHH05 boomUTR		map
	HaloTag	pCS2+ Halo-NbVHH05 boomUTR		map
	fbxw11b	pCS2+ VHH05grad		map
127d01	EGFP	pCS2+ EGFP-Nb127d01 boomUTR		map
	mNeonGreen	pCS2+ mNG-Nb127d01 boomUTR		map
	mScarlet-I	pCS2+ mScarleti-Nb127d01 boomUTR		map
	mTagBFP2	pCS2+ mTagBFP2-Nb127d01 boomUTR		map
	HaloTag	pCS2+ Halo-Nb127d01 boomUTR		map
	fbxw11b	pCS2+ 127d01grad		map
Sun	EGFP	pCS2+ EGFP-FbSun boomUTR		map
	fbxw11b	pCS2+ Sungrad		map
Moon	EGFP	pCS2+ EGFP-NbMoon boomUTR		map
	fbxw11b	pCS2+ Moongrad		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Unpublished GEARs Constructs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