CRISPR/Cas9 Technology & Genome Editing
At ingenious, we will work with you to design and customize your genetically engineered animal model(s) to meet your exact specifications using our CRISPR-Cas 9 technologies. The ingenious process for generating your custom mouse model is broken down into 3 steps:
1st Step: Strategy Design
Selection of target site, assays, and oligonucleotides, as well as predicted off-target sites. Preparation of sgRNA and Cas9 mRNA.
2nd Step: Production of Founder Mice
Injection of sgRNA and Cas9 mRNA into fertilized embryos, to generate F0 mutated candidate animals. Breeding and genotyping for the presence of the desired mutated candidate F0 animals.
3rd Step: Breeding of Founders to obtain F1 mice
We will breed the new founders to wildtype mice of matching strain background, and genotype their offspring to obtain F1 mice bearing the knockout or knockin allele. Optional breeding will be offered to segregate off-target mutations if necessary.
CRISPR-Assisted Targeting in ES Cells
Mouse knockouts and knockins can be generated by utilizing CRISPR-Assisted homologous recombination in ES cells. CRISPR-Assisted targeting projects provide the advantage of screening cells instead of mice, while at the same time reducing timelines and costs. This targeting provides the speed of CRISPR with the reliability of projects screened in ES cells.
The ingenious process for generating gene knockouts and/or knockins utilizing both CRISPR-Cas9 and ES cells is broken down into the following steps:
1st Step: Project Strategy Consultation and Design
Determining the characteristics of the target gene/locus. Selection of target site, assays, and oligonucleotides, and donor targeting vector. Preparation of sgRNA Cas9 mRNA, and donor targeting vector.
2nd Step: Electroporation and Tissue Culture using FLP ES cells
Your sgRNA and donor targeting vector will be sequenced and electroporated into our proprietary highly germline competent FLP ES cells. This technology alleviates the need for breeding out the Neo, which in turn saves 3-6 months of production time.
3rd Step: Screening for Positive Clones
Screening for Neo-negative targeted ES clones by PCR. After the initial positive clones are identified, the positive clones will be expanded and further confirmed via RT-PCR.
4th Step: Generation of Heterozygous Mice
Introduction of CRISPR-Assisted targeted ES cells into host embryos, followed by transfer into surrogate mothers. The resulting CRISPR Cas/9-Assisted targeted Neo-negative chimeras are mated with wildtype mice to obtain germline confirmed F1 mice.