CRISPR/Cas9-mediated Endogenous Fluorescent Tagging of Germline-specific Genes in Caenorhabditis elegans

Summary

A CRISPR/Cas9 microinjection workflow for endogenous fluorescent tagging in the Caenorhabditis elegans germline to obtain homozygous knock-in lines for in vivo protein localization analysis.

Abstract

Protein localization in the Caenorhabditis elegans (C. elegans) germline is central to interpreting gene function during gametogenesis, yet conventional transgene approaches often yield variable expression and can be silenced in germ cells. Here, a practical CRISPR/Cas9 workflow inserting a fluorescent tag into an endogenous locus is described, enabling the generation of stable knock-in alleles that report protein distribution under native regulation. The protocol covers key stages of the procedure: selecting a tagging strategy appropriate for the target protein, delivering CRISPR reagents by gonadal microinjection into young adult hermaphrodites, and recovering injected animals for screening. Knock-in candidates are identified through PCR-based genotyping across two generations to isolate homozygous worms and verify the edited allele. Finally, confocal microscopy is used to verify germline fluorescence and assess subcellular localization in vivo. The workflow is designed to be reproducible and broadly applicable to germline-enriched genes, providing a straightforward route to establish homozygous tagged strains for developmental and cell-biological analyses.

Introduction

Caenorhabditis elegans (C. elegans) is a well-established model for studying reproductive development and gametogenesis, owing to its short life cycle, transparent body, and high genetic tractability¹. C. elegans exists primarily as self-fertilizing hermaphrodites, whose gonad first undergoes spermatogenesis, and then switches to oogenesis. This reproductive mode simplifies strain maintenance and makes it straightforward to obtain homozygous lines². Recent spatial transcriptomics has substantially expanded the list of genes enriched in the gonad and has revealed pronounced sex-specific expressi....

Protocol

All experiments involving Caenorhabditis elegans were conducted in accordance with the institutional guidelines for the care and use of laboratory organisms at Beijing Normal University.

Figure 1: Overview of CRISPR/Cas9-mediated endogenous fluorescent tagging in C. elegans. Schematic workflow ....

Results

Following the protocol, young adult hermaphrodites were gonadally microinjected with a CRISPR/Cas9 plasmid (pDD162), a plasmid donor encoding a GFP knock-in tag flanked by about 1 kb homology arms (pPD95.75), and co-injection markers (pRF4 and pPD122.11). A germline-enriched target gene was used as a representative example to generate a stable, homozygous knock-in allele that reports protein localization under native regulatory control.

Across two injection sessions, 30 P0 hermaphrodites were .......

Discussion

This workflow is designed to generate stable, homozygous knock-in lines in which a fluorescent tag is inserted at the endogenous locus to report protein localization under native regulatory control^5,6,7,8,9,10. In practice, success is best judged by a sequential validation chain rather than any single readout: identification .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Beijing Lablead Biotech Co., Ltd.	QZ02	Component for preparing NGM agar plates
Agarose	NOVON	ZZ14011	Prepare 2% agarose pads
Caenorhabditis elegans strain N2	Caenorhabditis Genetics Center (CGC)		Injection background strain
Centrifuge	Eppendorf	5425	Clarify injection mix
Cholesterol	Beijing Chemical Reagents Company	57-88-5	Component for preparing NGM agar plates
Co-injection marker plasmid rol-6 (su1006) (pRF4)	Guangshuo Ou Lab		Roller marker to enrich candidates
Confocal microscope	ZEISS	LSM880 + Airyscan	Validate GFP expression/localization
Di-Potassium hydrogen phosphate (K2HPO4)	Sinapharm	CAS: 7758-11-4	Component for preparing NGM agar plates
Di-Sodium hydrogen phosphate (Na2HPO4)	Sinapharm	CAS: 7558-79-4	Component for preparing M9 buffer
DNA ladder	Mei5 Biotechnology, Co., Ltd.	MF288-01	Size reference for agarose gels
Donor plasmid backbone (pPD95.75)	Addgene	1494	Backbone for HDR donor construction
Electrophoresis  System	Beijing Liuyi Biotechnology Co., Ltd.	112-0630	Separate PCR amplicons on agarose gels for junction screening and zygosity genotyping
Escherichia coli OP50 strain	Caenorhabditis Genetics Center (CGC)		Food source for worms
Fluorescent co-injection marker plasmid (pPD122.11)	Fire Lab		GFP co-marker to enrich candidates
Fluorescent stereomicroscope	SOPTOP	SZX12-HT	Screen live worms for fluorescent co-injection markers
GelRed Nucleic Acid Gel Stain	Mei5 Biotechnology, Co., Ltd.	MF079-plus-01	Visualization of DNA bands
Glass capillaries	World Precision Instruments	1B100F-4	Used with needle puller to make injection needles
Halocarbon oil 700	Sigma-Aldrich	H8898-50ML	Prevent desiccation during injection
Incubator	Wuhan Ruihua Instrument & Equipment Co., Ltd.	HP400S	Maintain worms at standard temperature
Magnesium sulfate (MgSO4)	Sinapharm	CAS: 7487-88-9	Component for preparing NGM agar plates and M9 buffer
Microcapillary pipettes	KIMBLE	71900-50	Heated and snapped to create loading tool
Microinjector unit	Eppendorf	FemtoJet 4i	Deliver controlled pressure pulses of injection mix into the distal gonad via a microinjection needle (foot-pedal triggered)
Microscope cover glasses	Fisherbrand	12545A 22×30-1	Prepare 2% agarose pads for mounting and immobilizing worms during microinjection; Open injection needle tips against the coverslip edge
Microscope for microinjection	ZEISS	Axio Observer.A1	Injection microscope
Needle puller	Sutter Instrument Company	Model P97	Pull glass capillaries into injection needles
PCR thermocycler	Bio-Rad	C1000 Touch Thermal Cycler	PCR amplification
pDD162 (Peft-3::Cas9 + Empty sgRNA)	Addgene	47549	Cas9 + sgRNA expression
Peptone	Gibco	308723	Component for preparing NGM agar plates
Potassium dihydrogen phosphate (KH2PO4)	Sinapharm	CAS: 7778-77-0	Component for preparing NGM agar plates and M9 buffer
Sodium chloride (NaCl)	Sinapharm	CAS: 7647-14-5	Component for preparing NGM agar plates and M9 buffer
Stereomicroscope	Motic	K400L	Pick and mount worms on agarose pads and perform post-injection recovery steps