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Caenorhabditis elegans (C. elegans) is a widely used model organism in genetic research due to its simple biology, transparent body, and ease of genetic manipulation. Its short life cycle and well-mapped genome make it ideal for high-throughput genetic screening and CRISPR-Cas9-based gene editing.
CRISPR-Cas9 allows precise genome modifications by introducing targeted mutations. In C. elegans, this is achieved by injecting plasmids carrying specific guide RNA (sgRNA) sequences and homologous recombination (HR) templates into the worm's gonads. Key materials include Cas9-sgRNA plasmids (pDD162) and co-injection markers. Researchers design and verify sgRNA sequences, construct plasmids, and screen progeny for successful knockouts or knockins.
Figure 1. Schematic of CRISPR-Cas9 genome editing in C. elegans. (Kim HM, et al., 2019)
1. Cas9-sgRNA plasmid (without targeting sequence): pDD162
2. Roller co-injection marker: pRF4 plasmid
3. Optional cherry co-injection marker: pCFJ90 plasmid
4. Site-directed mutagenesis kit.
5. DNA purification.
6. DNA quantification: NanoDrop spectrophotometer
1. Design sgRNA guide sequences
a. Design at least two highly specific sgRNA targeting sequences for each knockout experiment.
b. Utilize the web-based sgRNA design tool.
c. Submit 50-200 bp genomic DNA sequence for each analysis (submit twice for longer sequence knockouts).
d. Settings: Sequence Type: other regions; Target genome: C. elegans (ce10)
Tips:
Choose guide sequences with higher scores (>90)
Avoid guide sequences with continuous T or high A-T base pairing
Avoid guide sequences prone to forming hairpins
2. Construct Cas9-sgRNA plasmid
a. Insert the desired targeting sequence into pDD162 using the site-directed mutagenesis kit.
b. PCR primers:
Forward primer (sgRNA-specific): 5'-N20GTTTTAGAGCTAGAAATAGCAAGT-3'(Replace N20 with the desired 20-bp targeting sequence)
Reverse primer (common for all sgRNAs): 5'-CAAGACATCTCGCAATAGG-3'
Note: The NGG motif must be present only in the genomic target sequence, not in the sgRNA sequence.
3. Confirm sgRNA plasmid by sequencing
Sequencing primer: 5'-GGTGTGAAATACCGCACAGA-3'
4. Design template plasmid for homologous recombination (HR)
a. Design 5' and 3' HR arm PCR primers according to the mutagenesis kit instructions.
b. Use 900-1200 bp HR arms for optimal results.
c. If sgRNA target sequences are present in the HR template plasmid, design mutagenesis primers to introduce silent mutations in either the PAM (NGG motif) or sgRNA targeting sequence.
Note: Verify the plasmid by sequencing before worm injection, ensuring correct translation frame, ATG start codon, and stop codon.
5. Purify and quantify plasmids
Use a NanoDrop spectrophotometer for concentration measurement.
6. Prepare injection mixture
| Component | Final Concentration |
| HR plasmid | 50 ng/μL |
| sgRNA-1 | 50 ng/μL |
| sgRNA-2 | 50 ng/μL |
| pRF4 | 50 ng/μL |
| pCFJ90 (optional) | 5 ng/μL |
| H2O | to final volume |
| Total volume | 5 μL |
7. Worm injection
a. Microinject the mixture into the gonads of young adult worms.
b. When F1 progeny reach L4 stage, isolate roller worms onto new OP50 plates.
c. Isolate 50-150 roller worms to obtain knockin worms.
8. Identify F1 heterozygous knock-in worms
a. Perform single-worm PCR using 2-2.5 μL worm lysate in a 10 μL PCR reaction.
b. Use knockin-specific primers for PCR amplification.
c. Confirm knockin worms by sequencing at this stage or in F2 generation if lysate is insufficient.
d. For site-directed mutation screening, digest 10 μL PCR product with the appropriate enzyme by adding 1/10 volume of 10x buffer and the desired enzyme.
9. Identify F2 homozygous knock-in worms
a. From KI-PCR positive F1 worms, isolate 10-20 F2 offspring onto new OP50 plates.
b. Culture until sufficient F3 worms hatch.
c. Identify homozygous knockin worms using both knockin-specific and wild-type primer pairs.
At Creative Biogene, we harness the transformative capabilities of CRISPR/Cas9 to bring unparalleled accuracy and efficiency to your research with Caenorhabditis elegans (C. elegans). Our cutting-edge CRISPR/Cas9 services are tailored to accelerate your projects and enhance your research outcomes.
Our Services Include:
Targeted Gene Mutations:
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