Home › CRISPR Model Creation › C. elegans Transgenic Services › C.elegans CRISPR Knockout
Knockouts are the bread and butter of reverse genetic studies. They allow researchers to study the effects of the absence of a particular gene, protein segment, amino acid, or regulatory element, thereby enabling the researcher to determine their function. Gene knockouts are a commonly used tool for biologists to understand gene function. Examination of phenotypes when the gene is deleted can reveal insights into what role that gene plays in the organism.
We work with you on project design using a collaborative approach to ensure that you receive the C. elegans line best suited for your experiments.
For research requiring a true null mutant, we recommend working with a precise KO to ensure the accuracy of your data. Many traditional null mutants rely on an early stop, which may not create a true null. Any genomic region can be targeted for deletion.
We can delete the entire coding sequence of a gene using CRISPR/Cas9. You can then study the phenotype of the true null allele with no concern that any protein function remains. Our transgenic designers and process ensure that each knockout we make is exactly the right strain to answer the research question.
The Precise Deletion service uses CRISPR/Cas9 genome editing to remove a small defined region of DNA. This can be useful for deleting protein domains, DNA regulatory regions, or any other sequence of your choice.
Deletions can also be performed in coding regions for functional analysis of protein domains or to isolate isoform function. Transcription factor binding sites or introns can be deleted to reveal information about gene regulation.
Floxed alleles can be created for a tissue- or temporal- specific deletion of your gene of interest. This is a powerful tool for studying embryonic lethal genes or for understanding how your gene of interest functions in different tissues. CRISPR/Cas9 is used to insert loxP sites flanking the region to be deleted.
This line can then be crossed with a Cre-line expressing the Cre recombinase under a specific promoter or injected with a plasmid containing Cre recombinase. The Cre recombinase promotes recombination of the two loxP sites and the region between the sites is removed from the genome.
We are able to build custom Cre recombinase-expressing lines to compliment your new floxed allele.
Case Study: Insertion of two loxP sites to produce a knockout (KO) of an embryonic lethal gene
A client wanted a knockout (KO) of an embryonic lethal gene. We could not make this line using our standard methods. Instead, we inserted two loxP sites. One in the first intron of the gene and the second in the 3’utr. After we confirmed this line by PCR and sequencing, we injected this line with a ubiquitously expressing Cre Recombinase plasmid.
We found while the uninjected animals could reproduce, the Cre injected animals did not (Figure A). We tested recombination by PCR and found that only the Cre injected progeny showed recombination of the loxP sites (Figure B).
This line could be used to study KO of this gene in adulthood or in specific tissues, something that was not previously possible due to the embryonic lethality caused by the KO of this gene.
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