Name: CRISPR/Cas9-mediated Endogenous Fluorescent Tagging of Germline-specific Genes in Caenorhabditis elegans (Video) | JoVE Journal
Uploaded: 2026-05-29T13:20:00.000+00:00
Duration: 10 min 37 s
Description: Learn CRISPR/Cas9 endogenous fluorescent tagging for germline protein localization in Caenorhabditis elegans. This protocol enables stable knock-in alleles with native regulatory control for accurate in vivo analysis.

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5 minutes

This work was supported by grants from the National Key Research and Development Program of China (2023YFA1801100 to L.M.), the Natural Science Foundation of China (32400698 to P.W., 32270774 to L.M.).

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.
<img alt="C. elegans CRISPR/Cas9 workflow diagram: gene editing, microinjection, sequence validation." class="xfigimg" src="/files/ftp_upload/70879/70879fig1.jpg" data-altupdatedat="1787025648000" data-alt="Figure 1"> 
Figure 1: Overview of CRISPR/Cas9-mediated endogenous fluorescent tagging in C. elegans. Schematic workflow illustrating the major stages of endogenous fluorescent knock-in generation and validation. In the design and construction stage, a germline-enriched target gene and insertion site are selected, pDD162 is used as the CRISPR/Cas9 plasmid for Cas9 and guide RNA expression, and pPD95.75 is used as the donor backbone into which the fluorescent tag (e.g., GFP) and the left and right homology arms are subcloned. In the microinjection stage, the CRISPR/Cas9 injection mixture (including the gRNA/Cas9 plasmid, donor template, and co-injection markers) is delivered into the gonad of young adult hermaphrodites, followed by recovery on seeded plates. In the validation stage, marker-positive progeny are selected for downstream genotyping, including PCR-based identification of knock-in events and confirmation by DNA sequencing, and successful knock-in lines are subsequently examined by fluorescence microscopy to assess germline expression and subcellular localization of the tagged protein. <a href="https://www.jove.com/files/ftp_upload/70879/70879fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Select a germline-enriched target gene
<ol>
	<li>Identify candidate genes with predominant expression in the gonad or germ cells using public databases and published literature.</li>
	<li>Select an appropriate tagging site based on protein features. Record the selected isoform and the tag insertion position for downstream guide RNA and donor template design (Figure 1).</li>
</ol>
2. Design the guide RNA and donor template
<ol>
	<li>Identify a 20 nt guide RNA (gRNA) sequence upstream of a protospacer adjacent motif (PAM; NGG), the sequence required for Cas9 recognition, and position it as close as possible to the intended tag insertion site.</li>
	<li>Evaluate candidate gRNAs using an online design tool (e.g., the IDT CRISPR design tool) with the C. elegans genome selected. Select a guide with strong predicted on-target cutting efficiency and no high-risk predicted off-target sites.</li>
	<li>Clone the gRNA sequence into the region between the U6 promoter and the gRNA scaffold within the CRISPR expression plasmid backbone. 
	NOTE: Design primers incorporating the gRNA sequence as overhangs. Perform PCR amplification followed by homologous recombination-based assembly, a ligation-independent cloning method that eliminates restriction enzyme digestion and enables one-step cloning directly into the expression vector15.</li>
	<li>Confirm correct insertion by Sanger sequencing.</li>
	<li>Construct the donor plasmid using a fluorescent tag flanked by approximately 1,000 bp left and 1,000 bp right homology arms derived from the target locus. 
	NOTE: Amplify homology arms from N2 genomic DNA prepared as worm lysate using proteinase K digestion. Design primers with 15–20 bp overlaps to the linearized vector and fluorescent tag. Assemble the homology arms and tag into the backbone via homologous recombination in a single reaction. Insert the fluorescent tag immediately after the ATG start codon for N-terminal tagging or before the stop codon for C-terminal tagging. Include a linker sequence between the target gene and the fluorescent tag to maintain proper protein folding and function. Use the linker to reduce steric interference between the fusion partners, and ensure that both the linker and tag are inserted in frame with the coding sequence. Select the insertion site carefully to avoid disrupting known or predicted functional domains, signal peptides, or transmembrane regions (Supplementary File 1).</li>
	<li>Introduce silent substitutions into the donor homologous template sequence to disrupt the PAM or guide-binding sequence without altering the protein sequence and prevent Cas9 re-cutting after integration. 
	NOTE: Incorporate synonymous point mutations into primers used for homology-arm amplification, targeting codons within the PAM region.</li>
	<li>Confirm donor sequence integrity across both homology arms and tag junctions by Sanger sequencing. 
	NOTE: Store verified plasmids at −20 °C for short-term storage or −80 °C for long-term storage.</li>
</ol>
3. Prepare the injection mix
<ol>
	<li>Assemble the injection mix by combining the CRISPR expression plasmid, the donor plasmid (constructed based on pPD95.75), and co-injection markers in sterile ddH₂O to a final volume of 10 µL at the concentrations listed in Table 1. 
	NOTE: Optimize concentrations empirically to balance marker readability, candidate recovery, and post-injection survival.</li>
	<li>Centrifuge the injection mix at 10,000 × g. for 10–20 min to pellet particulate material and prevent clogging of the microinjection capillary.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Function</td>
			<td>Working concentration (ng/μL)</td>
		</tr>
		<tr>
			<td>pDD162</td>
			<td>CRISPR/Cas9 plasmid (Cas9 and gRNA expression)</td>
			<td>50-100</td>
		</tr>
		<tr>
			<td>Donor plasmid (pPD95.75 backbone)</td>
			<td>HDR donor template carrying fluorescent tag flanked by ~1 kb homology arms</td>
			<td>50-100</td>
		</tr>
		<tr>
			<td>pRF4</td>
			<td>Co-injection marker (Roller phenotype)</td>
			<td>50-100</td>
		</tr>
		<tr>
			<td>pPD122.11</td>
			<td>Co-injection marker (GFP expression)</td>
			<td>5-10</td>
		</tr>
		<tr>
			<td>Sterile ddH2O</td>
			<td>Bring to final volume</td>
			<td>–</td>
		</tr>
	</tbody>
</table>
Table 1: Injection mix composition and plasmid functions. The injection mix contains CRISPR expression plasmid, donor plasmid, pRF4, and pPD122.11 at the indicated concentrations, for a total volume of 10 µL.
4. Load and prepare the injection needle
<ol>
	<li>Pull glass capillaries into microinjection needles using a needle puller configured to produce a long taper with a sharp tip suitable for gonadal injection. 
	NOTE: Optimize puller parameters empirically. Example starting parameters: heat = 540, pull = 20, velocity = 100. 
	CAUTION: Handle pulled needles carefully to avoid puncture injuries. Use forceps when handling needles and dispose of used needles in a sharps container.</li>
	<li>Heat a microcapillary pipette and snap it into two pieces to create a loading tool. 
	NOTE: Commercial loading tips may be used as an alternative.</li>
	<li>Attach a rubber aspirator bulb to the wide end of the shortened microcapillary pipette.</li>
	<li>Aspirate approximately 1 µL of clarified injection mix supernatant into the loader. 
	NOTE: Exact volume is not critical. Ensure the solution fills the needle tip.</li>
	<li>Insert the loader into the back end of the injection needle and gently dispense the injection mix.</li>
	<li>Place the loaded needle horizontally for 10 min to allow the solution to settle toward the tip and confirm a continuous column.</li>
	<li>Mount the needle on the injection microscope and bring the tip into focus using brightfield optics.</li>
	<li>Open the needle tip by gently contacting it with the edge of a coverslip in halocarbon oil and test flow. 
	NOTE: Adjust injection pressure (30–80 psi) to generate a bubble approximately equal to the gonad diameter within 1 s16.</li>
</ol>
5. Prepare worms for injection
NOTE: Prepare Nematode Growth Medium (NGM) plates and M9 buffer as described previously17. Maintain worms on plates seeded with Escherichia coli OP50 (hereafter OP50) and perform injections in the N2 background.
<ol>
	<li>Select L4 hermaphrodites from a healthy plate and incubate at 20 °C for 10 h to obtain young adults with well-developed germlines.</li>
	<li>Transfer worms to an unseeded plate to reduce bacterial carryover.</li>
	<li>Prepare a dry 2% agarose pad on a glass slide and apply a drop of halocarbon oil.</li>
	<li>Transfer a worm onto the agarose pad using an eyelash picker.</li>
	<li>Immobilize the worm and orient the gonad for injection. 
	CAUTION: Complete injections within 20–30 min to prevent desiccation.</li>
</ol>
6. Perform gonadal microinjection
<img alt="C. elegans diagram showing germline region with labeled sections, microscopy research in genetics." class="xfigimg" src="/files/ftp_upload/70879/70879fig2.jpg" data-altupdatedat="1787025648000" data-alt="Figure 2"> 
Figure 2: Localization of the distal gonad syncytium for microinjection in young adult C. elegans hermaphrodites. (A) Low-magnification brightfield image of a mounted young adult hermaphrodite. Both gonad arms are visible; the injection site is indicated for one arm (arrow) as a representative example. The boxed region marks the area shown at higher magnification in panel B. (B) Higher-magnification DIC view of the distal gonad corresponding to the boxed region in panel A. The arrow indicates the injection position within the distal gonadal syncytium, where the injection mix is delivered. Scale bars: 100 µm (A) and 20 µm (B). <a href="https://www.jove.com/files/ftp_upload/70879/70879fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Locate an immobilized worm and identify the gonad using a low-magnification objective.</li>
	<li>Position the needle using micromanipulator controls.</li>
	<li>Orient the worm at a 20–40° angle relative to the needle.</li>
	<li>Focus on the distal germline using higher magnification.</li>
	<li>Advance the worm toward the needle and position the tip within the gonadal syncytium (Figure 2).</li>
	<li>Inject the solution and confirm delivery by visible gonad swelling. 
	NOTE: Optimize injection pressure (30–80 psi), compensation pressure (0.1–0.5 psi), and injection duration (0.1–0.3 s). 
	CAUTION: Excessive pressure or volume can cause worm rupture.</li>
	<li>Withdraw the needle and reposition for subsequent injections. 
	NOTE: Perform a unilateral injection to maximize survival, or a bilateral injection to increase editing efficiency.</li>
	<li>Add M9 buffer to release worms and transfer each worm to an individual recovery plate.</li>
	<li>Repeat injections for 20–25 worms. 
	NOTE: Replace the needle if it clogs or becomes damaged.</li>
	<li>Incubate injected worms at 20 °C for 2–3 days to allow recovery.</li>
</ol>
7. Screen F1 candidates to identify knock-in-positive lines
<ol>
	<li>Screen F1 progeny for co-injection markers and select marker-positive animals. 
	NOTE: Examples of alternative fluorescent co-injection markers with different tissue specificity and spectral properties are provided in Supplementary Table 1. 
	NOTE: Select animals expressing both markers for higher reliability. Prioritize broods with high marker frequency (“jackpot broods”).</li>
	<li>Transfer each F1 worm to an individual plate and allow egg laying for 1–2 days.</li>
	<li>Lyse each F1 worm in proteinase K buffer to prepare PCR templates. 
	NOTE: Store lysates at −80 °C if not processed immediately.</li>
	<li>Perform junction PCR using one genomic primer outside the homology arm and one internal tag primer. 
	NOTE: Retain corresponding plates from PCR-positive F1 animals.</li>
</ol>
8. Screen F2 progeny to isolate homozygous knock-in lines and confirm integration
<ol>
	<li>Isolate F2 progeny from PCR-positive F1 lines onto individual plates.</li>
	<li>Allow F2 worms to produce progeny and lyse adults for genotyping. 
	NOTE: Store lysates at −20 °C if needed.</li>
	<li>Perform PCR using primers flanking the homology arms to distinguish wild-type and knock-in alleles by product size.</li>
	<li>Confirm correct integration by Sanger sequencing across both insertion junctions.</li>
	<li>Verify germline fluorescence expression and localization using fluorescence microscopy.</li>
	<li>Expand confirmed homozygous lines and maintain stocks. 
	NOTE: Cryopreserve validated lines for long-term storage.</li>
</ol>

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The authors have nothing to disclose.

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 control5,6,7,8,9,10. In practice, success is best judged by a sequential validation chain rather than any single readout: identification ...

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 expression patterns across the reproductive system in C. elegans3. More recently, single-cell RNA sequencing has enabled reconstruction of germ-cell developmental trajectories and inference of gene regulatory networks governing spermatogenesis4. Together, these datasets generate rich candidate lists, but functional interpretation often requires observing protein behavior in vivo&#8212;specifically, defining when and where the corresponding proteins localize and how their patterns change throughout germline development.
Conventional transgenesis in C. elegans often relies on gonadal microinjection of plasmid DNA, which readily generates multi-copy extrachromosomal arrays5. However, in the germline, these arrays are often robustly silenced, a mechanism that is thought to help maintain germline integrity by restricting expression from repetitive DNA, thereby preserving germline function and the stability of genetic information across generations6,7. As a result, transgene expression constructs that are readily expressed in somatic tissues can be weak or inconsistent in the germline. Even when expression is detectable, copy-number effects and engineered regulatory elements can shift protein dosage and timing, potentially confounding conclusions about native localization and dynamics. To address these limitations, microparticle bombardment and Mos1-mediated Single Copy Insertion (MosSCI) are widely used to generate low/single copy integrated transgenic lines8,9,10. These approaches can improve expression in the germline, yet they differ in cost, throughput, and how well they reproduce endogenous regulation. CRISPR/Cas9 genome editing offers a more direct route by inserting a fluorescent tag at the endogenous locus via&#160;homology-directed repair, generating stable knock-in alleles that report protein localization and dynamics under native regulatory control11,12,13,14.
Here, a practical workflow for CRISPR/Cas9-mediated endogenous fluorescent tagging in C. elegans is described, applied to genes selected based on reported germline-enriched expression patterns. The protocol outlines target selection, donor template and guide RNA design, gonadal microinjection, and downstream screening to isolate homozygous knock-in lines, followed by validation using genotyping. This approach is broadly applicable to germline-expressed genes across developmental stages and enables quantitative analyses of protein localization and dynamics in vivo. This workflow provides a practical strategy for tagging genes expressed in the germ line and for analyzing protein localization and dynamics in vivo&#160;under native regulatory control.

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

crispr/cas9-mediated endogenous fluorescent tagging of germline-specific genes in caenorhabditis elegans

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

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.

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 ...

A CRISPR/Cas9 microinjection workflow for endogenous fluorescent tagging in the Caenorhabditis elegans germline to obtain homozygous knock-in lines for in vivo&#160;protein localization analysis.

Protein localization in the Caenorhabditis elegans (C. elegans) germline is central to interpreting gene function during gametogenesis, yet conventional ...

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.

crisprcas9-mediated-endogenous-fluorescent-tagging-germline-specific

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Biology

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The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

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CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

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Learn CRISPR/Cas9 endogenous fluorescent tagging for germline protein localization in Caenorhabditis elegans. This protocol enables stable knock-in alleles with native regulatory control for accurate in vivo analysis.

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

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