InDel Mutations by CRISPR/Cas9


A method that markedly accelerates the generation of genetically modified mice involves the CRISPR/Cas9 nuclease system, which facilitates the generation of DNA double-strand breaks (DSBs) at spec­ified genomic loci, which are then repaired by error-prone non-homologous end joining (NHEJ), resulting in mutant alleles. 


This procedure currently requires the delivery of Cas9 protein, along with single-guide RNA (sgRNA), both of which cooperatively produce sequence-specific DSBs, into one-cell embryos i.e. zygotes. Synthetic sgRNAs are nowadays most frequently purchased commercially. Delivery is achieved by a) Electroporation, b) Standard Pronuclear Injection (PNI), or c) Piezo-Assisted Cytoplasmic Injection.


Our Core can assist in selecting the sgRNAs with the best-predicted efficiency and specificity, before they are ordered commercially. More than one sgRNAs are selected and ordered for each locus. The cutting efficiency of those is then tested in a Zygote Assay, whereby the sgRNAs are delivered into a small number of embryos, the embryos are grown in vitro till the blastocyst stage and blastocysts are sequenced and analyzed for incidence of cutting. The sgRNA with the best cutting score is then selected for the next round at which point the surviving embryos are transferred into the oviduct of pseudopregnant females, resulting in pups (potentially mosaic) for knock-out mutations via InDel formation.


Pups are typically transferred to the investigator for analysis and subsequent mating to the F1 generation to establish the mutation in the germline. At roughly 10 days of age, a tail biopsy is obtained from each pup and is given to the investigator lab for genotyping. Our facility kindly requests results in a timely fashion. Upon identification of mutant pups, a transfer process (involving a pathogen test) to the investigator lab is initiated.








External Academic


Assistance with Project Design




Purchasing of synthetic sgRNA




InDel (guarantee: 1 founder or 20 pups)




Additional Costs




$2 cage/day

Additional Injections After Deliverables Reached




Other Costs (Tail biopsies, Pathogen tests, shipping to external labs, etc)









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