How do I prep my CRISPR-Cas9 reagents for micro-injection and/or transfection? Please refer to the Technical Bulletin for Micro-injection Ready Reagents
My prepped RNA is sticky/viscous/contains debris/ too many embryos are lysing when I try to inject. How can I fix this? First try enlarging the opening of your needle (i.e. break the tip against the holding pipette). Second, if that does not help spin the sample down in the MI ready vial at 10,000 rpm (or top speed) for 10 minutes. Use only the top layer to load a new needle. Try spinning and reloading at least two times.Third, proceed to a new Micro-injection ready aliquot if issues persist. Repeat steps 1 and 2 with new aliquot if issues remain.
How many validated sgRNAs do I receive when I purchase the Validated or Validated+ option? You receive 1 validated sgRNA. The validation process consists of testing up to 5 sgRNAs and the most active sgRNA is prepared and sent to you. If more than 1 sgRNA is found to be active, the additional sgRNA(s) can be purchased for $200 each.
I do not know how to genotype my animals that have been born using the microinjection-ready reagents. Validated genotyping protocols are provided on all Validated and Validated+ Certificates of Analysis documents upon receipt of your reagents. Please reference these documents for specific genotyping instructions and suggestions. These protocols provide genotyping instructions and details to identify your desired mutation. The protocols include primer sequences and PCR parameters. Below are suggestions and troubleshooting for identifying your founder animals:
It is recommended to sequence verify the 5’jxn and 3’jxn PCR products for knockin positive animals.
The NHEJ PCR assay can also be sequence analyzed for sgRNA cutting activity.
For best results, it is recommended that the PCR products undergo a purification step to remove residual dNTPsand primers prior to sequencing.
For knock-in projects, It does not matter whether or not the animal contains mutations from NHEJ, as it typically is on a different allele and will separate out at the heterozygous generation when the founder is backcrossed to wildtype. This PCR assesses CRISPR-Cas9 efficiency during founder screening, and will be used for wildtype(WT) allele amplification to differentiate between heterozygous versus homozygous knockin animals.
For knock-in projects, it is possible to see both random and targeted integration in the same animal. Depending on where the random integration occurred, it may or may not separate out at the heterozygous generation when the founder is backcrossed to wildtype.
Horizon also offers a genotyping service. You can send your samples to our labs to be genotyped by our scientists to alleviate your workload.
My birthrates are low, what should I do? The typical birthrate is 10% (Pups born/embryos transferred) for both rats and mice. Some models require screening of 50 to 100 pups and require increasing the number of micro-injections performed If you are not detecting knockin events, first thing to check is CRISPR-Cas9 activity (refer to NHEJ PCR in provided genotyping protocol)
Below are questions to ask yourself to help determine the issue:
Is your gene of interest embryonic lethal?
Is your gene of interest X-linked?
If this is a knockin model, does the insertion of the donor cassette interfere with the endogenous gene function that could cause lethality?
Where are you injecting your CRISPR-Cas9 reagents? Pronucleus or cytoplasm? We recommend pronucleus.
Are you injecting multiple sgRNAs or oligo donors at one time? Injecting multiple sgRNAs and oligo donors can have a negative effect on birthrate. If you see this happen we suggest diluting the oligo donors before mixing with the sgRNA/Cas9.
How do you address off target effects? While there is always a chance for off-targeting (OTE) to occur, Horizon has established stringent guidelines for our design process. The sgRNAs are designed using an algorithm to generate only sgRNAs with the highest design score for your region of interest. Each design is blasted against the top 10 most homologous sites in a given genome. Horizon has conducted several screens testing for OTE in multiple models, and have not found any off target effects to date.
Horizon has been using a mRNA delivery system for micro-injection in embryos for the past 5 years. By using mRNA to deliver CRISPR-Cas9 into an embryo, we have the flexibility to optimize concentrations to minimize the reagent’s exposure time in the cells. We use the lowest concentration of Cas9 mRNA and sgRNA that can be injected into an embryo while obtaining maximum modification efficiency.
Furthermore, by monitoring breeding schemes, any unwanted mutations can be bred out of a colony. While the chance of OTE exists, the possibility a founder animal will contain an OTE at one of the top 10 potential sites is low. If an OTE were to occur in a founder animal, the unwanted mutation should separate at the next generation, when backcrossed to wildtype.