Zebrafish Knock-in Services

Zebrafish Knock-in Services

Zebrafish is gaining popularity as a model organism for human genetic disease research. Point mutation knock-in using CRISPR/Cas9 allows scientists to create genetic models using zebrafish for understanding gene function, basic biology, and more precisely modeling human diseases.

What is Zebrafish Knock-in?

Knock-Ins in zebrafish consist of inserting a specific sequence of DNA at a specific site in the zebrafish genome.  The two most popular gene-editing methods used are CRISPR/Cas9 and Tol2.

A wide array of molecular tools at researchers' disposal, as well as an increasing number of published examples of knock-ins in the literature, make knock-ins seem relatively straightforward. However, engineering these precise modifications remains a challenge for many researchers.

Keys to a Successful Knock-in in Zebrafish

  1. Know your gene - Good knowledge of your gene, alternate isoforms expressed and related orthologs is required for designing an efficient experimental strategy.
  2. Choose good sgRNAs - To ensure you use an sgRNA that guides efficient cutting, 2-3 sgRNAs with different recognition and PAM sites should be designed for each knock-in experiment 
  3. Validate sgRNAs - All sgRNAs should be validated in vivo to ensure that they are not toxic and that they efficiently guide Cas9 cutting of the DNA.
    Read our article on why validation is important.
  4. Create the right donor DNA template - The size of your knock-in insert determines the type of template (double-stranded vs. single-stranded) and the size of the homology arms used.
  5. Design and test primers - Developing a robust assay to detect your edit is critical when you begin screening the embryos produced by the F0 founders.
  6. Screen, Screen, Screen - Knock-ins are notoriously low efficiency, and on top of that, the F0 germline is mosaic and can transmit multiple edits. The rate of transmission to your F1 generation will vary depending on when the F0 germline edit was made in development. Ranges from 3% to 50% have been observed. Be sure to grow enough F1 animals that you will be able to find your F1 heterozygotes. 

Download the infographic: 6 Essential steps to a successful zebrafish knock-in project. 

Download the infographic: Using zebrafish for rapid genetic analysis. 

Knock-in Techniques:

  • Knocking in point mutations. Specific coding sequences can be changed to precisely match genetic variants seen in human disease.
  • Knocking in a fluorescent tag at the native locus. Observe when and where your gene of interest is expressed using fluorescence. A genetically encoded fluorescent marker (GFP, mCherry, etc.) can be inserted in-frame for tagging of either the N- or C-terminus of your protein.
  • Knocking in a protein tag. Genetically encoded tags (FLAG tag, HA-tag, poly(His) tag, etc.) can be inserted in-frame for tagging of either the N- or C-terminus of your protein. The protein can then be observed by Western blots, immunofluorescence, and immunoprecipitation using commercially available common antibodies.
  • Knocking in LoxP sites. Insertion of two LoxP sites flanking your gene of interest creates the ability to eliminate the gene of interest with expression of the Cre recombinase. Cre recombinase can be expressed using tissue or stage-specific promoters for precise control of gene function.

Key References

A thorough review of current methods around point mutation knock-ins in zebrafish using CRISPR/Cas9, including the detailed description of the screening workflow for identifying the rare precise editing events generated with current knock-in approaches.

Download the publication.

An excellent overview of knock-ins in zebrafish. The article provides one of the most comprehensive side-by-side comparisons of donor template designs and knock-in strategies available for zebrafish at the time. A detailed discussion of the different repair pathways, template designs, and different reagent choices, as well as their limitations and paths forward for improving knock-in editing efficiencies in the future is very informative

Download the publication.

Boel et al. delve into the dizzying realm of ssODN-mediated knock-in repair with a comprehensive assessment of the impacts of different repair templates and design strategies on editing outcomes. Potential mechanisms of repair that lead to complex mutation patterns obtained with ssODN templates, as well as potential avenues for improvement on these methods are also discussed.

Download the publication.

Our Capabilities

The InVivo Biosystems scientific team has 40 years of combined zebrafish gene editing experience. With access to the state-of-art University of Oregon Zebrafish Facility and top-rated zebrafish researchers on campus, our team has successfully introduced point mutations by knocking in sequence variants to study gene function. 

With our ability to generate zebrafish knock-in lines, we can: 

  • Model human genetic diseases by knocking in point mutations
  • Track gene expression and protein localization by knocking in a fluorescent tag at the native locus.
  • Add immunohistochemistry to your toolbox by knocking in a protein tag.
  • Create conditional control of gene function by knocking in LoxP sites. (See figure below).

 

Schematic of a PCR gel testing 4 samples for a loxP insertion.  When loxP is successfully inserted, DNA fragments in the amplification region are ~50bp longer. This difference can be seen on a gel, and founders transmitting this edit can be identified to establish a stable line.

Knock-in Offerings (price reflects academic pricing)

Knock-in PackagesPoint Mutation Fluorescent Tag Protein TagLoxP
PriceDeliveryPriceDeliveryPriceDeliveryPriceDelivery
Full Build$26,9959-12 months$27,8359-12 months$27,2409-12 months$27,36518 months
Verified Clutch*$9,3104-6 months$10,1905-6 months$9,5955-6 monthsN/AN/A
Custom Injection Mix
(with verification of sgRNA cutting)
$3,2594-8 weeks$5,259 & up6-8 weeks$5,259 & up6-8 weeks$4,2554-8 weeks
Custom Injection Mix
(without verification of sgRNA cutting)
$9954-6 weeks$2,995 & up4-6 weeks$2,995 & up
4-6 weeks$1,9902-4 weeks
*Verified Clutch service is only available to customers in the United States.
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