Small Fragment & Point 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 Homologous DNA Repair mechanisms. 

 

The generation of Point Mutation Mice or Mice with Small Inserts 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. In addition, for small fragment insertions and point mutations, an oligo donor carrying the desired mutation and homology arms is co-delivered to zygotes. Synthetic sgRNAs are purchased commercially and so is a single stranded DNA donor oligo, which usually consists of a point-mutation site, flanked by 60 base sequences on each side adjoining the DSBs. 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. Similarly, we can provide guidance for the design of ssDNA oligos, which are also ordered commercially. More than one sgRNAs are selected and ordered for each locus. The cutting efficiency of those is first 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 involving delivery of the ssDNA, in addition to Cas9 and sgRNA. The surviving embryos are transferred into the oviduct of pseudopregnant females, resulting in mutant pups (potentially mosaic).

 

Pups are typically transferred to the investigator lab 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.

 



FEES

 

WI

MIT

External Academic

 

Assistance with Project Design

FREE

$100/h

$120/h

Purchasing of synthetic sgRNA

~$160/sgRNA

~$160/sgRNA

~$160/sgRNA

Zygote Test for sgRNA Validation

$750

$1,500

$2,000

Point Mutation – BDF1 background (guarantee: 1 founder or 20 pups)

$4,000

$5,000

$6,000

Point Mutation – C57Bl/6 background (guarantee: 1 founder or 20 pups)

$4,750

$5,750

$6,750

Additional Costs

Housing

$1.5 cage/day

$1.5 cage/day

$2 cage/day

Additional Injections After Deliverables Reached

$750/day

$1,000

$1,500

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

variable

variable

variable