Before You Begin: Design and Genotyping
- Design: We strongly recommend that we are involved in the design of the targeting strategy. The efficiency and thus the likelihood of success depends critically on the design. Mistakes in mice are expensive in terms of both money and time.
- Genotyping: Design of a genotyping strategy is an integral part of the design process--another reason to involve us in the design process. The mice we give you will be useless if you can't identify which carry the desired mutation.
- No passenger mutations: An additional consideration is that some of the repair pathways engaged by CRISPR/Cas are error-prone, in particular those engaged when single-stranded oligos are used to incorporate missense mutations. In these cases, we recommend careful characterization of the targeted gene in the progeny of founder mice mated to wild type, to ensure that there are no second-site mutations in the same gene at a distance from the intended change.
CRISPR/Cas Reagents For Injection
- Reagents for injection are commercially prepared. Contact us for details.
- The current commercial vendor costs for injection-ready reagents (guide RNA, Cas9 protein and ssDNA oligo or dsDNA targeting vector) are from $1000 to $2000.
CRISPR/Cas Mutagenesis by Injection of Fertilized Eggs
What We Will Do Part I
What You Will Provide Part I
- We will advise you. Early in your planning, please consult with Ron Conlon and/or David LePage to discuss your project.
What We Will Do Part II
- An account number.
- We require a demonstration that the guide RNA(s) directs sequence-specific cutting in an in vitro assay.
What You Will Provide Part II
- We will inject fertilized mouse eggs with the reagents.
- While we don't provide a guarantee, our goal is to deliver enough mice to generate the desired mutant mice.
- We will house the mice until they are safe to transport.
- You will receive and house the mice.
- You will genotype the mice we provide, and tell us which mice and how many are mutant.
- We ask that you acknowledge the contribution of the Case Transgenic and Targeting Facility in seminars and publications.
- Investigators need an IACUC protocol from their institution to receive mice from the core. CWRU IACUC protocols need to specify use of the Case Transgenic Core. Investigators normally do not need an IBC protocol of their own for this work: the review for recombinant DNA in animals will be conducted on the information submitted through online ordering for the transgenic core, and will be added to the core's IBC protocol as an amendment if approved. You may be contacted by the IBC for additional information as part of rDNA review after submission of the order. IBC approval is required prior to initiation of injections.
- Currently (12/1/22), the core is injecting 4 weeks after reagents are available and the project is ready. During this time mice to fulfill the service are ordered and received from Jax.
- From the injection date, it will be a minimum of a further 6 weeks before we will be able to deliver mice to you (almost 3 weeks for gestation, and at least 3 weeks of postnatal growth before weaning).
- $5,000 and the cost of donor females for CRISPR/Cas9 on the B6SJL background. The donor female cost ranges from $1,000-$2,000.
- $6,000 and the cost of donor females for CRISPR/Cas9 on C57Bl/6J inbred background. The donor female cost ranges from $1,000-$2,000.
- Local, non-CWRU orders will be charged an additional $200 for packing and transporting mice. Non-local orders will be charged $200 and the costs of transportation.
CRISPR Mutant Mouse Ordering
You will need the following information to order
As well as
- PI Name, Address and Contact Info
- Account Number
- IACUC Protocol Number
- Project name
- Mutation Type
- Mouse Strain to Inject
- Is a Phenotype expected?
- Building and Room to Deliver Mice to
(for rDNA/IBC approval
- Purpose and design of the animal experiments
- Lay description of the animal experiments
- Name and function of the gene
- Whether the gene is involved in infectious disease in normal healthy adult humans
- Whether the gene presents any hazard to health or the environment
- A .pdf file diagram the targeting strategy
CRISPR/Cas Success Rates
We have completed about 100 different CRISPR knockin/out projects. The vast majority have generated multiple independent founders with the desired allele. Typically founder mice are not mosaic for the founder allele, unlike in early applications of this technology. We have successfully generated many knockout, point mutation knockins, reporter/tag/Cre knockins, floxed or conditional alleles, Rosa26 and H11 safe harbor targeted transgenes, and floxed-stop alleles.
Practical Guidance for CRISPR/Cas Design
- Point mutations should be made with a single guide RNA and the nuclease version of Cas9. We strongly recommend against the strategy using two offset gRNAs and Cas9 nickase. The three priorities in successful knockin design are 1) a gRNA that cuts close (10bp or less) to the desired mutation 2) validation that the gRNA is active and 3) mutations that stop continued cutting through mutation of the PAM or gRNA seed.
- For knockouts, we recommend using two gRNAs and Cas9 nuclease to delete an essential exon. Reliance on indels can result in non-null alleles. Moreover, a large, designed deletion is easier to detect by genotyping.
- We will help you identify candidate guide RNAs for your mutation. If potential off-target sites are not on the same chromosome as your gene, any off-target mutations will segregate away through meiosis when you breed the mice. It will also be preferable if the off-target sites are in sites biologically unrelated to your target (for example, not in a coding sequence of a related gene).
- We will ask you to identify which candidate gRNAs are active with an in vitro cutting assay. gRNAs generated with the kit are assayed by incubation with Cas9 protein provided by the kit and your amplified genomic target, then running the products on an agarose gel like a restriction nuclease assay. Run uncut target genomic fragment in one lane and the different gRNA assays on the same gel to determine which gRNAs most efficiently direct cutting--the majority are active, you want to avoid the ones which don't work. In our experience, this in vitro cleavage assay correlates better with gene targeting in injected eggs than the predictions of online tools or cell transfection assays.
- For knockins, once an active gRNA which cuts close the desired mutation (ideally 10bp or less) has been identified, design silent passenger mutations that disrupt the PAM (preferred) or gRNA seed (the 10 nucleotides closest to the PAM) to prevent recutting of recombinants. If your substitution generates a mutation in the PAM or seed sequence, it may not be necessary to incorporate passenger mutations. When designing synonymous passenger mutations, be certain to avoid rarely used codons using a mammalian codon bias table. It is possible to recover desired edits without destroying the PAM or seed sequence, but the yield can be much lower because of recutting and mutagenesis of already recombined alleles. Single-stranded oligos of 100bp centered on the mutation are sufficiently long for substitution mutations.
- Purchase the reagents. We purchase our reagents (guide RNAs and Cas9 protein and PAGE-purified oligo) for injection of fertilized eggs from a commercial vendor.
- You will genotype the founder mice using the assays that we develop during the design phase.
- Validate the mutation by sequencing the locus carefully in F1 mice (the offspring of founder mice mated to wild type mice). There have been reports that some of the mutations made can be complex at the targeted locus. This characterization cannot be performed easily in the founders because the founder mice could carry different mutations--you may be unable to tell if different mutations are in the same cell or on the same chromosome.
- Treat each founder mouse as the start of an individual mutant line. You should characterize (at least preliminarily) multiple independent mutations. The standards are still being set in this field, and it may become a requirement to show that multiple, independent mutations have the same phenotype, in order to account for potential linked, off-target mutations. There has been the suggestion that two independently generated mutations should be analyzed.