Before You Begin: Design and Genotyping

CRISPR/Cas Reagents For Injection

CRISPR/Cas Mutagenesis by Injection of Fertilized Eggs

What We Will Do Part I What You Will Provide Part I What We Will Do Part II What You Will Provide Part II Acknowledgements Regulatory Compliance Timeline Cost

CRISPR Mutant Mouse Ordering

You will need the following information to order
  • 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
As well as (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

  1. 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.
  2. 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.
  3. 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).
  4. 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.
  5. 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.
  6. 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.
  7. You will genotype the founder mice using the assays that we develop during the design phase.
  8. 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.
  9. 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.