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

We use CRISPR-based endogenous tagging to study the localization and dynamic of germline proteins in C.elegans. Conventional transgenes may affect the germline expression, but this protocol tag proteins at the endogenous locus under native control. To begin, identify the candidate genes with predominant expression in the gonad or germ cells using public databases and published literature.

After opening the UniProt entry for the target protein, click Structure to evaluate structural features and protein domains and identify an appropriate fluorescent tagging site. Scroll down and open the AlphaFold DB entry to verify the model confidence values. Click on Domains for specific structural classifications.

Next, open the target gene page in WormBase. Scroll down and click on Sequences widget, and the primary transcript link to open the transcript page. Scroll down to the Sequences section and click on the specific option to reveal the spliced sequence of the selected transcript.

In the IDT CRISPA-Cas9 Guide RNA Design Tool webpage, copy and paste the first exon sequence of the target gene from the WormBase page, and select C, elegans from the Species dropdown menu for guide RNA or gRNA design. Click on the DESIGN button to obtain and review the top recommended gRNA sequence and its corresponding PAM sequence. On SnapGene, select Sequence to identify the cloning point between the CeU6 promoter and the gRNA scaffold regions.

Copy the gRNA sequence from the IDT tool page, paste it in the insertion dialogue box, and click Insert to integrate the spacer into the CRISPR expression plasmid. To confirm the accuracy of gRNA insertion, select the sequence and click on Show alignments to compare the Sanger sequencing result base by base with the expected sequence. To construct the donor plasmid, copy the homology arm's genomic sequences from WormBase.

In SnapGene, paste the sequence and click Insert. Next, right-click the insertion site to open the Set DNA Color window and change it to Orange to mark the junction. Copy the green fluorescent protein or GFP insert sequence and paste it directly at the marked position.

Then click Add Feature. Name the feature GFP. Change its display color to green and click OK to complete the in silico donor plasmid assembly.

To introduce a silent substitution into the donor plasmid template for preventing Cas9 recutting after integration, copy the 19-nucleotide gRNA spacer from the expression vector. In the donor plasmid tab, paste the sequence into the Find DNA sequence search bar to locate the target site and select the specific nucleotide position to mutate. In Replace base, enter new nucleotide and click Insert.

Right click the modified base and use Set DNA Color to change its color to red. Align the donor plasmid Sanger sequencing results with the expected sequence to confirm integrity. Prepare the injection mix by combining the CRISPR expression plasmid, the donor plasmid, and co-injection markers in sterile double distilled water to a final volume of 10 microliters at the concentration shown.

Then centrifuge the mix at 10, 000 g for 10 to 20 minutes to pellet out particulate material and prevent clogging of the microinjection capillary. Use a needle puller to create microinjection needles from glass capillaries with a long taper and sharp tip suitable for gonadal injection, while optimizing the polar settings empirically. Heat a microcapillary pipette and snap it into two pieces to create a loading tool.

Attach a rubber aspirator bulb to the wide end of the shortened microcapillary pipette. Aspirate approximately one microliter of injection mix into the loader. Insert the loader into the back end of the injection needle and gently dispense the injection mix.

Place the loaded needle horizontally for 10 minutes to form a continuous column and allow the solution to settle toward the tip. Mount the needle on the injection microscope and bring the tip into focus using bright-field optics. Open the needle tip by gentle contact with the edge of a cover slip in halocarbon oil and verify flow.

To culture the worms for injection, maintain the N2 wild-type worms on OP50 bacteria-seeded media plates. Select hermaphrodites at the L4 stage from a healthy plate and incubate at 20 degrees Celsius for 10 hours to obtain young adults with well-developed germlines. Transfer worms to an unseeded plate to reduce bacterial carryover.

Prepare a dry 2%agarose pad on a glass slide. And apply a drop of halocarbon oil. Use an eyelash picker to transfer a worm onto the agarose pad and immobilize it.

Use a low-magnification objective to orient the gonad for injection. Use micromanipulator controls to position the needle at a 20 to 40-degree angle relative to the worm. Use high magnification to focus on the distal germline.

Advance the worm onto the needle and position the tip within the gonadal syncytium. Inject the solution until the gonad swells. Withdraw the needle and reposition for subsequent injections.

Add M9 buffer to soften and partially break up the agarose pad to release worms. Transfer each worm to an individual recovery plate. Then repeat injections for 20 to 25 worms.

Screen F1 progeny for co-injection markers and select marker-positive animals. Transfer each F1 worm to an individual plate and allow for one to two days for egg laying. Lyse each F1 worm in proteinase K buffer to prepare PCR templates.

Perform junction PCR using one genomic primer outside the homology arm and one internal tag primer. And retain corresponding plates from PCR-positive F1 animals. Isolate F2 progeny from PCR-positive F1 lines onto individual plates to produce progeny.

Lyse adults for genotyping. Perform PCR using primers flanking the homology arms to distinguish wild-type and knock-in alleles by product size. Use the SnapGene tool to verify and validate correct integration at both the left and right insertion junctions by aligning Sanger sequencing results with the expected edited genomic sequence.

Use fluorescence microscopy to verify germline expression and localization. Expand confirmed homozygous lines. Maintain stocks and cryopreserve validated lines for long-term storage.

PCR-based screening of F1 progeny revealed successful knock-ins identified by 1, 550-base-pairs junction PCR amplicon using one flanking genomic and one internal GFP primer. The absence of the 1, 550-base-pairs band in most F1 animals indicated no detectable target integration, consistent with non-integrative DNA transmission rather than homology-directed repair. Zygosity determination in F2 progeny derived from junction PCR-positive lines distinguishes alleles by band size at 1, 345 base pairs as wild-type allele, and 2, 344 base pairs as knock-in allele.

Heterozygotes show both bands. Homozygous knock-in samples lack the wild-type-sized band, and the samples with failed integration show only the wild-type-sized product. Additional validation of the homozygous GFP knock-in line was done using confocal microscopic imaging, where GFP fluorescence in the germline cells with signal enriched in the spermatogenic region was observed.

This protocol enables in vivo analysis of protein localization and dynamics in the C.elegans germline. Careful tag design and thorough validation are essential to preserve protein function and minimize false-positive candidates. Future studies can use this workflow to track protein localization and explore potential interactions during germline development.