Zebrafish Floxed Gene Services
CRISPR/Cas9 is used to insert loxP sites flanking the region to be deleted in zebrafish. Insertion of two loxP sites creates the ability to permanently remove the sequences of interest with expression of the Cre recombinase which can be expressed using tissue or stage-specific promoters for precise control of gene function.
This line can then be crossed with a Cre-line expressing the Cre recombinase under a specific promoter or injected with a plasmid containing Cre recombinase. The Cre recombinase promotes recombination of the two loxP sites and the region between the sites is removed from the genome.
We can create a floxed allele for tissue-specific deletion or temporal-control of your gene of interest. This can be very useful when studying embryonic lethal genes or for understanding how your gene of interest functions in different tissues.
Conditional Mutagenesis by loxP Insertion
One of the more difficult aspects of biology is how to perform loss-of-function studies of essential genes. Now the conditional mutagenesis by loxP insertion in zebrafish is possible.
We use sequential integration of loxP sequences (e.g. two stages of injection and screening) to create "floxed" genes for large deletions of sequence in the targeted loci.
Conditional alleles
- Used to make deletion of genes in cell type and time specific manner
- Flank gene of interest with loxP sites
- Inject a plasmid or cross into strain that expresses Cre recombinase where/when/how you want loss-of-function to occur
Conditional mutagenesis by loxP insertion in zebrafish – outfoxing the unfloxable
By Ben Jussila
Floxed Gene Service Options
| Standard Injection Mix | Standard Injection Mix + Screening Primers | Validated sgRNA Injection Mix | Complete Validated Injection Mix | Full Build* (Sequence verified heterozygous line) | |
| Floxed Gene 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* | |||||
| Timeline | 4-6 weeks | 4-8 weeks | 1-2 months | 4-6 months | n/a |
| More Details | More Details | More Details | More Details | More Details |
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
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?
- Verified 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
Injection Mix
Injection Mix
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.
- Custom reagents from validated suppliers
- Four 10uL lyophilized injection mixes with instructions on reconstitution
- Rhodamine dye for early visualization of injection success
Screening Reagents
Screening Reagents
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.
- PCR primers designed and tested to identify the target edit
- PCR protocol to identify animals that contain your edit
sgRNA Testing
sgRNA Testing
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.
- Up to 3 sgRNAs tested for CRISPR cutting efficiency in zebrafish embryos
- Selection against sgRNAs that have toxic effects
- Identification of an sgRNA that is successful for guiding CRISPR cutting
In Vivo Editing Efficiency Screening
In Vivo Editing Efficiency Screening
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.
- Test injection of mix into embryos
- Integration efficiency in injected embryos
- Sequencing to confirm precise edit
Expertly Injected Embryos
Expertly Injected Embryos
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.
Germline Transmission Screening & Line Propagation
Germline Transmission Screening & Line Propagation
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.
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