zebrafish CRISPR Knock-In

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Zebrafish CRISPR Knock-in Services

Zebrafish is gaining popularity as a model organism for human genetic disease research. Knock-ins in zebrafish consist of inserting a specific sequence of DNA at a specific site in the zebrafish genome. 

Point mutation knock-in using CRISPR/Cas9 enables researchers to create zebrafish genetic models in order to understand gene function, basic biology, and more precisely model human diseases.

Our scientists can help you design your next disease model to your precise specifications using CRISPR techniques. Our expertise can help you:

  • Improve your success rate in generating lines
  • Avoid repeating mistakes and reduce variabilities

Knock-in Techniques

Knock-in is a powerful tool to test the function of specific domains in a protein or to model known human mutations in research animals. These small precise changes in specific amino acid sequence can be used to better understand how gene mutations are associated with human health and disease.

We can successfully repair a point mutation by knocking in a wild-type repair template to restore gene function. Below is a visual readout of knock-in repair in F0 (injected, generation zero) zebrafish larvae carrying a point mutation in a gene essential for pigmentation. To determine if this repair has been transmitted through the germline, we would subsequently raise these F0 animals to adulthood and screen their progeny for wild-type development of pigment cells. Images were taken at 48 hours post-fertilization (hpf).

nacre 48 hpf: a nacre mutant larvae, homozygous loss of function mutation in the gene mitfa (microphthalmia-associated transcription factor a) causing a loss of melanophores (pigment cells)
wt 48 hpf : a wild type larvae with normal development and distribution of melanophores.
DN rescue 48 hpf: nacre mutant embryos injected with Cas9 + gRNA + ssODN (single stranded oligodeoxynucleotides) showing repair of the mitfa mutation, resulting in rescued development of melanophores to varying degrees (highest to lowest degree of rescue, top to bottom)

Knock-in techniques in zebrafish utilize CRISPR/Cas9 to insert small sequence changes at a specific site in the zebrafish genome. sgRNA site(s) are selected to open the genome adjacent to the desired edit, and a repair template is used to introduce precise changes in the genome. Animals are then raised to adulthood and screened for transmission of the desired edit.

  • 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. Learn more>>.

(Left): SelN-BFPSTOP with BFP followed by a STOP codon inserted into the N-terminus of Selenon (SelN). (Center and Right): Ryr1a-mCherry line where the endogenous Ryr1a was tagged with mCherry at the N-terminus. Image courtesy of Melissa A. Wright, MD/PhD, Assistant Professor of Pediatric Neurology at University of Colorado. Read the customer story.

Modeling STXBP1 Patient Variants in Zebrafish

Illustration of a successful precise point mutation of stxbp 1a in zebrafish. stxbp1a is a highly conserved zebrafish ortholog of human STXBP1 (87% identity). Using CRISPR/Cas9 technology, we were able to precisely generate a benign patient mutation at the conserved amino acid residue (CCC>CTG, p.P94L).

Keys to a Successful Knock-in in Zebrafish

  • Know your gene – Good knowledge of your gene, alternate isoforms expressed and related orthologs is required for designing an efficient experimental strategy.
  • 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
  • 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.
  • 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.
  • 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.
  • 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. 

6 Essential steps to a successful zebrafish knock-in project.

Using zebrafish for rapid genetic analysis.

CRISPR Knock-in Service Options

Standard Injection Mix

Validated sgRNA Injection Mix

Fully Validated Injection Mix

Mosaic Clutch* (F0 injected embryos)

Full Build** (Sequence verified heterozygous line)

KI Design

Injection Mix Assembly

Locus Evaluation & Sequencing Primers

in vivo sgRNA Testing

in vivo Editing Assessment & Screening Reagents

Expertly Injected Embryos

Germline Transmission Screening & Line Propagation

* This service is only available to clients in the United States.
** Full Build includes screening n=100 adults; standard husbandry charge applies.

Injection Mix and sgRNA Validation Details

Standard Injection Mix

Choose this option if you want us to pick your sgRNA site, have already tested your sgRNA in vivo, or want to make a CRISPR knockout. We send a custom designed injection mix and you develop your own preferred detection/screening protocol.

What’s Included

  • Consultation with a genetic engineer to develop the genome editing strategy
  • In silico design of all reagents including the sgRNA site and donor homology template
  • Sequence files for the donor homology construct and edited locus
  • 4 vials of 10uL lyophilized sgRNAs duplexed with the Cas9 protein
  • Injection dyes for early visualization of injection success
  • Detailed Instructions on mix reconstitution and injection protocols.

Standard Injection Mix + Screening Primers

Choose this option if you want to inject and screen for your edit by PCR without having to worry about designing your own screening primers. This option includes our standard mix plus ready-to-go screening primers.

What’s Included?

  • Prescreened Allele Specific PCR primers to detect your edit in zebrafish tissue.

Complete Validated Injection Mix

Choose this option if you want a CRISPR knockin with completely validated reagents to use in your lab and to find your gene edited line. This is the best Custom Injection Mix option for the novice zebrafish line builder.

What’s Included?

  • Full in vivo evaluation of somatic editing efficiency.
  • Confirmation of integration capacity of your edit into the zebrafish genome

Mosaic Clutch (F0 injected embryos)

Choose this option if you want a CRISPR knock-in with completely validated reagents to use in your lab and to find your gene edited line. This is a good option for the researcher who wants to outsource part of the workload in the development of a new line.

What’s Included?

  • Full in vivo evaluation of somatic editing efficiency.
  • Confirmation of integration capacity of your edit into the zebrafish genome

Validated sgRNA Injection Mix

Choose this option if you want us to guarantee your injection mix is designed around a high efficiency sgRNA site using our rigorous in-house testing process.

What’s Included?

  • Expertly injected embryos for direct submission to you nursery
  • Fully validated PCR primers and protocols for edit detection assay in your own lab.

Full Build (Sequence verified heterozygous line)

Choose this option if you want us to deliver a stable sequence validated CRISPR knock-in line.

What’s Included?

  • Growth of F0 injected embryos in our nursery
  • Rigous screening of F0 injected adults for germline transmission of your edit
  • Sequence conformation and propagation of F1 heterozygous animals

Injection Mix Service Component Descriptions

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.


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