Conditional Knock Out 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 Mice with Conditional Alleles 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, one or more donor template(s), in the form of single stranded DNA (ssDNA) or a double stranded DNA plasmid is/are co-delivered to zygotes. Synthetic sgRNAs and ssDNAs (in cases where sequence allows) are purchased commercially. Plasmids are provided by the investigator lab. Delivery is achieved by a) Standard Pronuclear Injection (PNI), or b) 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 some guidance/resources for the design of the donor DNA. More than one sgRNAs are selected and ordered for each locus. The cutting efficiency of those is first analyzed 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

CKO – BDF1 background (guarantee: 1 founder or 50 pups)

$5,500

$8,000

$10,000

CKO – C57Bl/6 background (guarantee: 1 founder or 50 pups)

$6,500

$9,000

$11,000

Additional Costs

Housing

$1.5/cage/day

$1.5cage/day

$2 cage/day

Additional Injections After Deliverables Reached

$1,000/day

$2,000

$2,500

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

variable

variable

variable