All the Power of CRISPR: Complete CRISPR Injection Mix & sgRNA Validation
CRISPR/Cas9 gene editing is a rapidly growing molecular biology toolset with diverse applications. However, designing and executing a CRISPR project turns out to be more complex than it appears from the outset. Reagent sourcing, and design and validation can be a time intensive process requiring strong internal expertise without a guarantee of success.
We know you have tight deadlines, and may have limited hands or expertise. Our goal is to help you get the tools you need to move forward with your experiments and accelerate the time from your idea to execution of the project. You can work with our team of genome editing specialists to design your injection mix so all you have to do is add water, inject and start screening!
Zebrafish Injection Mix: Comparing Buy vs. DIY.
Is CRISPR Injection Mix the right choice for you?
Our CRISPR injection mixes are fully customizable to get you the edit you want along with the support you need to start your project. Our 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. Our Standard Mix is for experienced researchers with extensive injection and screening competencies; the Complete Validated Mix is a rigorously tested injection mix for researchers looking for a fully plug and play format.
To mitigate failure rates, our custom injection mix includes an expertly designed set of reagents and tools that can help you:
- Build tools without the hassle. We put all the moving parts together for you to create the consistency in sourcing and design of the reagents.
- Improve success rate. Our custom injection mix packages incorporate sgRNA validation/cutting efficiency to mitigate failure rates.
- Enable do-it-yourself. Our team will do the heavy lifting of the project design and reagent procurement so you can take advantage of CRISPR/Cas9 technology.
- Save time. You can eliminate lengthy and variable workflow leading up to experiment start time.
- Jumpstart your project. You select how much help you need without having to be a CRISPR expert.
Images below show two different zebrafish lines using the CRISPR Injection Mix. (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.
Injection Mix and sgRNA Validation Offerings
Looking for an easy way to bring CRISPR gene editing into your lab? Choose the level of service that is appropriate for you.
|Standard Injection Mix||Standard Injection Mix + Screening Primers||Validated sgRNA Injection Mix||Complete Validated Injection Mix|
|In vivo sgRNA Testing|
|In vivo Editing Assesment|
|Timeline||4 weeks||6 weeks||8-10 weeks||12+ weeks|
|More Details||More Details||More Details||More Details|
*Pricing is for point mutation and knockout projects. Projects that require plasmid builds adds $2,000 and 4-6 weeks to each project.
*In-depth consultation with our expert CRISPR designers and editors can be included with any project.
What do I need?
In order to use our injection mix, you must have access to:
- Microinjection rig and capabilities for early embryo injection.
- Facilities for zebrafish growth, maintenance, and crossing.
- PCR machines and reagents.
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.
- 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.
- 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.
- 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.
- 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
Injection Mix Service Component Descriptions
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
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
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 Assesment
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