Design Construct Deliver Custom Gene-Edited Zebrafish
We are a zebrafish CRO that offers multiple gene-editing services including but not limited to mutagenesis, tagging, and transgenesis using CRISPR and Tol2 genome editing tools.
Our scientific team has 40 years of combined zebrafish gene editing experience. Our services can significantly reduce your experiment design time and can be tailored to different experimental requirements.
How To Choose Our Services
Our goal is help you move forward with your experiments and accelerate the time from a project idea to execution of the project. Select what works the best for you.
I am new to zebrafish genome editing
- I have an experimental idea, but not sure how to proceed
- I know the data I’d like to get, but am not sure what tool to use
- I’d like to talk to an expert
I work with zebrafish, but need help
- Injection & In Vivo testing
- Identify a founder/Screening
- Selecting quality reagents
Types of Edits
We offer a variety of service packages to fit the needs and budget of your lab. Choices range from the fast, affordable Custom Injection Mix to the comprehensive Full Build service packages, allowing you to customize the support you need to accomplish your gene editing goals.
We work with you on project design using a collaborative approach to ensure that you receive the zebrafish line best suited for your experiments.
We offer a variety of service packages to fit the needs and budget of your lab. Our services are fully customizable to get you the edit you want along with the support you need to start your project.
- CRISPR Injection Mix: Our Custom Injection Mix packages range from standard ready-to-inject mixes to full in vivo evaluation of sgRNA cutting efficiency and in-house validated screening tools to accommodate YOUR level of expertise. Learn more.
- Verified Clutch (F0 Injected Embryos): We develop and rigorously test your custom injection mix and deliver expertly injected embryos ready to submit to your nursery.
- Full Build (Sequence Verified Heterozygous Line): Our full service package handles the entire process of design, validation, injection, rearing, and germline screening, to deliver stable transgenic zebrafish lines.
The InVivo Biosystems Workflow
InVivo Biosystems provides a streamlined process for zebrafish gene editing services.
Donor Homology Design
- Project Design:
Selection of best guide RNAs surrounding the locus to be edited.
- sgRNA Testing:
In vivo cutting assayed by PCR/sequencing to determine the most efficient sgRNA.
- Donor Homology Design:
Repair templates are designed around high quality sgRNAs.
Skilled injectors for rigorous in vivo testing of editing reagents.
- Clutch Verification:
Evaluation of edit by PCR and/or sequencing of 10% of injected embryos.
- Germline Verification:
Verification of germline editing.
Comparison of Zebrafish Genome Editing Methods
|Nature of Edit||Number of Insertions||Germline Edits||Germline Efficiency||Common Terminology||Phenotype in F0 (injected embryos)||Max. Size of Insert|
|Morpholinos||Yes, but transient expression is variable, depends on quality of MO|
- Tol2: Most commonly used in zebrafish labs to make fast and rapid genome edits by random insertion of reporter cassettes (e.g. GFP, overexpression of mutant proteins). However, this method is not locus specific. It does not always recapitulate native expression patterns of gene and does not produce disruption in WT sequence. InVivo Biosystems does not used the Tol2 method to produce mutants.
- CRISPR/Cas9: Most commonly used to “Knock out” by INDEL (insertion/deletion around a double stranded break), “Knock in” point mutations (SDM; Site Directed Mutagensis) or Tags into native locus. A powerful genome editing method but it is more labor and time intensive to generate stable lines (germline). Compared to Tol2, CRISPR/Cas9 is less efficient but much more precise. F0 animals are called “Crispants”. These edits are highly mosaic in F0s which makes phenotyping challenging.
- Morpholinos (MOs): Most commonly used to get a functional “knockdown” of gene expression in F0 animals via small synthetic oligonucleotides that block translation or splicing of mRNAs. F0 animals called “Morphants”. Phenotyping is possible with good controls but declining concentration of MO leads to sub optimal knock down usually by 4dpf. Quick and very easy way to see if your GOI might have a phenotype and warrant making a germline mutant/null.
The InVivo Biosystems scientific team has 40 years of combined zebrafish gene editing experience. With access to the state-of-the-art University of Oregon Zebrafish Facility and world-renowned 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. Schematic created using BioRender.
Frequently Asked Questions
First, you need to inform us of your gene of interest so we can provide you with information regarding how we will be able to assist. Second, you need to choose a service package of interest. It can be full build, verified clutch, or custom injection mix. Depending on your choice, the timeline varies.
Complete Injection Mix is simple to work with, all you need to do is add water, inject, and screen. Our Complete CRISPR Injection Mix includes Cas9 protein, an RNA-based sgRNA(s), and a donor homology construct and can be shipped as a lyophilized mix in as little as 2 weeks. Discuss your research goals with us, and NemaMetrix will provide you with an expertly designed injection mix to generate your new zebrafish line.
Using Tol2 transgenesis you can generate Cre Recombinase-expressing lines, transcription reporters, models to analyze tissue-specific gene expression, models to control the timing of gene expression, and humanized zebrafish. Insertion content can range between 2 – 10 kb.
Live animal shipment depends on each individual country’s import regulations & guidelines. We can ship embryos or adult fish as long as they are allowed in the receiving country.
Having very high similarity in genetic makeup with humans, zebrafish has proved to be an important model for human genetics and disease. By using zebrafish knockout or knock-in models, genes can be manipulated to understand different disease pathways.
Yes, we can help you visualize protein expression using fluorescent protein tagging and epitope tagging.
• In zebrafish, fluorescent proteins are added using CRISPR/Cas9 gene editing technology. You can choose from a variety of proteins including YFP, BFP, GFP, and mCherry.
• Epitope tags in zebrafish are also added using CRISPR/Cas9 gene editing technology. The addition of an epitope tag at the endogenous locus enables you to quantify your protein without altering the level of gene expression. You can choose from a variety of tags including FLAG, HA, HIS, TAP, or S-peptide.
Custom tags and combinations (i.e. GFP::3xFLAG, etc) are also available.
Verification of the edit is performed by PCR and/or sequencing of a selection of injected embryos.
Using CRISPR/Cas9 technology, we can modify the zebrafish genome through Knockout, Point Mutation, Precise Deletion, Floxed Allele, etc. Specific experiment requirements can be met by working with our expert design team members.
Prices stated are exclusive of all taxes, fees, licenses, duties or levies and, unless otherwise stated in the quotation, transportation charges, freight and insurance.
For Verified Clutch and Full Build services you will be billed on a 3 step payment plan: 30% to initiate Phase I, 30% to initiate Phase II, 40% is due upon project completion and delivery.
For all other project types (e.g., Custom Injection Mix, and evaluation of sgRNA cutting), full payment will be invoiced and due prior to shipment of the project, unless otherwise set forth in the Quote.
Every reasonable effort will be made to identify the desired germline edit. In the event the edit is not identified a husbandry charge will still be assessed. The client will not be liable for the remainder of the Phase III cost.
Yes, we do. In case you could not find what you are looking for. Please feel free to contact us at [email protected].
Our scientific team has 40 years of combined zebrafish gene editing experience and access to the start-of-art University of Oregon Zebrafish Facility and top-rated zebrafish researchers. We use CRISPR and Tol2 as our genome editing methods. Our service can significantly reduce your experiment design time and can be tailored to different experimental requirements.
A well rounded review provides a detailed roadmap outlining -numerous tools available for genetic modification in zebrafish, including the historical and experimental context in which they have been used. Methods including Tol2 transgenesis, TALENs and CRISPR/Cas9 are discussed. A particularly useful flowchart can help readers understand the numerous decision points and considerations involved in choosing the right approach for their particular experimental needs.
An excellent framework for the novice and expert genome engineer alike.The article outlines the design of a number of elegant genetic tools to facilitate CRISPR/Cas9 mediated knock-ins, as well as downstream screening and experimental uses. The focus here is on fluorescent tags, epitope tags, conditional tools, and transgenesis markers to help researchers screen for phenotypically silent edits.
If you’re looking for more mechanistic insight into gene editing in zebrafish, look no further than this review from Simone et al. The authors provide a detailed discussion of the different DNA repair pathways, template types, and experimental considerations that all factor into a successful gene editing endeavor. The various “designer nucleases” available for zebrafish gene editing applications, their advantages, and their limitations are also discussed.