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.
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FEES |
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WI |
MIT |
External Academic |
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Assistance
with Project Design |
FREE |
$100/h |
$120/h |
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Purchasing
of synthetic sgRNA |
~$160/sgRNA |
~$160/sgRNA |
~$160/sgRNA |
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InDel (guarantee: 1 founder or 20 pups) |
$3,500 |
$4,500 |
$5,000 |
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Additional Costs |
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Housing |
$1.5/cage/day |
$1.5cage/day |
$2
cage/day |
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Additional
Injections After Deliverables Reached |
$750/day |
$1,000 |
$1,500 |
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Other
Costs (Tail biopsies, Pathogen tests, shipping to external labs, etc) |
variable |
variable |
variable |
