Mutant mouse models derived using engineered endonuclease technologies, like CRISPR/Cas9, bypasses the need for gene targeting in embryonic stem (ES) cells by directly manipulating the genome in mouse zygotes. Using this approach, the time for generating knockout, knockin, conditional, and targeted transgenic lines is reduced from many (>12) months with traditional gene targeting in ES cells to just a few (17-23) weeks with CRISPR/Cas9.
The following services are included in knockout (indel) mouse model creation by CRISPR/Cas9 targeting. Additional services are included upon request for mutant lines (e.g., conditionals, knockins, targeted transgenics) generated using homology directed repair (HDR) and homologous recombination (HR).
The MBP will perform a thorough informatics review of the gene of interest and provide a full design schematic illustrating the target allele, critical exon, and guide RNA (gRNA) sequence(s). For construct validation, the MBP will perform an in vitro cleavage assay to validate targeting efficiency of the proposed gRNA expression constructs. Briefly, genomic DNA encompassing the gRNA(s) binding site will be amplified by PCR. The resulting amplicon will be incubated with gRNA(s) and Cas9 (nuclease) and analyzed for expected fragments via agarose gel electrophoresis. This is an essential quality control step to verify gRNA targeting for Cas9-induced double stranded break (DSB) prior to microinjection. Only gRNA(s) that pass this quality control step will be used for microinjection.
Microinjection/Electroporation service includes manipulation of up to 180 fertilized C57BL/6N zygotes (other strains available upon request), embryo transfers into pseudopregnant recipients, and housing of pups for up to 10 weeks of age. Embryo transfer is intended to generate at least 1 genotypically-confirmed F0 (“founder”).
Genomic DNA isolated from tail or toe snips collected from 10-12 day old born pups will be used for PCR amplification of the endogenous loci followed by sequencing. Sequence chromatograms will show mixed sequences at the cut site, including sequence repaired by non-homologous endjoining (NHEJ). Each PCR product with mixed sequencing is sub-cloned. Up to 10 individual clones will be isolated and sequenced to confirm targeting.
Off-target nuclease activity is potentially problematic using Cas9. Although not verified, off-target editing can theoretically lead to unintended consequences that might obscure phenotypes associated with functional mutation of the intended target gene. For this reason, off-target analysis is highly recommended and encouraged. The MBP will use informatics tools (e.g., NCBI Blast, CRISPR.mit.edu) to identify the 10 most likely exonic off-target sites associated with the gRNA(s) sequences. In genotypically-confirmed F0 mice, potential off-target loci will be analyzed by PCR amplification and sequencing. If found, off-target mutations will be monitored to ensure segregation from the intended genetic mutation and elimination from subsequent generations of offspring. Results of off-target analysis will be provided to the client as information to guide further breeding and genotyping.
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Lee AY, Lloyd KC. Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice. FEBS Open Bio. 2014 Jul 1;4:637-42. doi: 10.1016/j.fob.2014.06.007. eCollection 2014. PubMed PMID: 25161872; PubMed Central PMCID: PMC4141200
Modzelewski AJ, Chen S, Willis BJ, Lloyd KCK, Wood JA, He L. Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc. 2018 13:1253-1274. Doi: 10.1038/nprot.2018.012