Zebrafish Knock-in Tagging Services

Tag your favorite gene at the native locus in your zebrafish line via CRISPR/Cas9 gene editing technology. Choose from a large selection of fluorescent proteins and epitope tags.
- Fluorescent tagging is typically used to aid in visualization of spatiotemporal dynamics of a given gene's protein product in live (and sometimes fixed) animals.
- Epitope tagging is most often utilized to facilitate biochemical studies, in particular for studies of protein-protein interactions which require the presence of a readily recognizable epitope or sequence feature for chemical modification or isolation.
Knock-in Tagging Techniques:
- 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.
Fluorescent Tagging vs. Epitope Tagging in Zebrafish
Fluorescent Tagging | Epitope Tagging | |
Example of tags | ||
Applications | in fixed tissue samples |
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Pros | Can aid in visualization of spatiotemporal dynamics of a given gene's protein product in live (and sometimes fixed) animals. | Can facilitate visualization or isolation of protein expression with reduced chance of disrupting endogenous expression levels or protein trafficking dynamics due to relatively small size. Can also facilitate visualization or biochemical modification of proteins in fixed tissue with comparatively robust and cheap commercially available antibodies. |
Cons | Can interfere with gene function due to the relatively large size of fluorophores used. | Not sufficiently validated. Lack of publications. |
(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.
Knock-in Tagging Service Options (price reflects academic pricing)
Standard Injection Mix | Standard Injection Mix + Screening Primers | Validated sgRNA Injection Mix | Complete Validated Injection Mix | Verified Clutch* (F0 injected Embryos) | Full Build** (Sequence verified heterozygous line) | |
KI Tag Design | ![]() | ![]() | ![]() | ![]() | ![]() | ![]() |
Injection Mix Assembly | ![]() | ![]() | ![]() | ![]() | ![]() | ![]() |
Locus Evaluation & Screening Reagents | ![]() | ![]() | ![]() | ![]() | ![]() |
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InVivo sgRNA Testing | ![]() | ![]() | ![]() | ![]() |
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InVivo Editing Assessment | ![]() | ![]() | ![]() |
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Expertly Injected Embryos | ![]() | ![]() |
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Germline Transmission Screening & Line Propagation | ![]() |
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Pricing | $2,995 | $4,595 | $5,995 | $7,995 | $11,395 | $28,995 |
Timeline | 4 - 6 weeks | 6 - 8 weeks | 8-10 weeks | 10 - 12 weeks | 4- 8 months | 9 - 12 months |
More Details | More Details | More Details | More Details | More Details | More Details |
* This service is only available to clients in the United States.
** Full Build includes screening 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?
- Our Standard Injection Mix PLUS:
- Prescreened Allele Specific PCR primers to detect your edit in zebrafish tissue.
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?
- Our Standard Injection Mix PLUS:
- Selection and testing of two sgRNAs for in vivo evaluation of cutting efficiency
- Donor homology designed around a demonstrated high efficiency sgRNA site
- In vivo screening for potential polymorphisms in your target locus
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?
- OUR VALIDATED sgRNA INJECTION MIX PLUS:
- Full in vivo evaluation of somatic editing efficiency.
- Confirmation of integration capacity of your edit into the zebrafish genome
Verified Clutch (F0 injected Embryos)
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 a good option for the researcher who wants to outsource part of the workload in the development of a new line.
What’s Included?
- Our Complete Validated Injection Mix PLUS:
- 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 knockin line.
What’s Included?
- Veried Clutch (F0 injected Embryos) Plus:
- 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.
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
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.
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