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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:
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).
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.
(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.
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).
* This service is only available to clients in the United States.
** Full Build includes screening n=100 adults; standard husbandry charge applies.
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
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?
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?
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?
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?
Choose this option if you want us to deliver a stable sequence validated CRISPR knock-in line.
What’s Included?
We create a mix using quality custom reagents from our validated suppliers. The sgRNAs are duplexed with the Cas9 protein.
The mix is created using concentrations of Cas9, sgRNA and donor homology that work well in our genome editing pipeline. The mix is provided as a stable dehydrated reagent.
Four injection mixes are provided that reconstitute to 5ul of mix each.
You can miss your edit if you don’t have a good assay to detect it.
We will design and test a robust assay to detect your edit, which is critical when you begin germline screening.
We will also provide PCR primers and protocols for an edit detection assay.
Ensure that you have the quality reagents you need for a successful genome edit. The efficiency of the sgRNA guided cutting can vary widely and is directly tied to the achievement of a successful genome edit.
Algorithms and experience help us choose the best candidate sgRNAs for your project, but the guided cutting efficiency of an sgRNA can be impacted by genome organization, for example, chromatin accessibility. The best way to determine the effectiveness of an sgRNA in a Zebrafish genome is to test it in vivo in the injected embryos.
We test your sgRNAs for their ability to guide Cas9 cutting. Our process also identifies SNPs and polymorphoisms in your locus that may impact homology directed repair. Embryos are injected with two different sgRNA/Cas9 duplexes and we determine the cutting efficiency for each sgRNA. If both sgRNAs fail to guide successful cutting, a third sgRNA is injected and tested.
Additional sgRNA testing can also be added with consultation.
Have total peace of mind by having us test your injection mix in our facility.
Our skilled injectionists will inject embryos with your mix and determine the somatic integration efficiency by PCR.
We will confirm the precise edit by sequencing to ensure that your edit is occurring correctly with no erroneous insertions.
Our team precisely injects the sg(s) targeting the locus or region of interest, Cas9 protein and the donor homology template to generate F0 embryos.
F0 embryos are reared to adulthood and then screened for germline transmission using a combination of screening techniques to identify germline founders. Identified adults are propagated to ensure a sufficient number of animals to ship to your facility.
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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An expert in CRISPR genome editing, InVivo Biosystems creates custom genome-edited C. elegans and zebrafish models to enable aging, developmental, and disease studies. Our unique in vivo platforms and technologies bridge the gap between cells and mice.
