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Research

Generation of Knock-in Mice Using Single-Stranded Oligodeoxynucleotides (ssODNs)

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General Description

Content

CRISPR/Cas9-initiated HDR in mouse embryos will be used to generate founder animals harboring inserted sequence (point mutations, epitope tags, loxP sites) or specific interval deletion alleles. At this time, the Core can perform genome editing with S.p. Cas9 or A.s. Cas12a/Cpf1. The targeting will be done in embryos using electroporation to introduce the CRISPR reagents.

What Will Happen

  1. The User Investigator will initiate a project in iLabs. An IACUC protocol number and account must be provided at this time.
  2. A consultation meeting between the Core and User Investigator will be held to discuss the desired genome modification. 
  3. Based on this discussion the Core will:
    • Design a genome editing approach to produce and detect the desired allele:
      • Point mutation alleles: With information from the user describing the desired point mutation, the Core will select guide RNAs, design a suitable ssODN repair template, and design a PCR-based genotyping scheme. The donor design will include silent mutations to modify the target sequence in the donor DNA, and possibly introduce a novel restriction site to facilitate genotyping. The overall design will be reviewed with the User and modifications made if necessary.
      • Epitope tag alleles: With information from the user describing the location of the epitope tag, the Core will select guide RNAs, design an ssODN repair template to insert the specified tag, and design a PCR-based genotyping scheme. The design will be reviewed with the User and modifications made if necessary.
      • Conditional alleles: The core will select a critical exon, select guide RNAs, design two ssODN repair templates to insert the loxP sequences, and design a PCR-based genotyping scheme. The design will be reviewed with the User and modifications made if necessary.
    • Review genome editing approaches designed by the User Investigator, including verification that the approach will produce the desired allele, independent computational assessment of guide RNA quality, and review of the genotyping approach. The requested information must be provided to the Core and the Core must approve the approach prior to proceeding to the next steps.
  4. The Core will have the guide RNA(s) synthesized by an approved vendor. Lab produced guide RNAs will not be used.
  5. Guide RNA(s) will be checked for concentration and degradation after resuspension.
  6. User investigators can opt to have the guide RNAs tested for genome editing efficiency in mouse zygotes prior to the production attempt [see Guide RNA testing in mouse zygotes].
  7. The Core will have the ssODN repair template(s) synthesized as custom Ultramers by IDT.
  8. The guide RNA(s) and Cas9 protein will be complexed into RNPs and an electroporation mix will be prepared with the ssODN repair template(s) in nuclease-free buffer at concentrations determined by the core.
  9. 200 C57BL/6J, C57BL/6N, or FVB/NJ embryos collected from superovulated females will be electroporated and transferred to pseudopregnant females. Please contact the GERM Core to discuss the necessary steps to use other strains.
  10. Live-born founder animals will be held by the GERM Core until 14 days of age and subsequently transferred to the User Investigator.
  11. Genotyping:
    • It is recommended that the Core perform genotyping of live-born offspring for the desired genome editing event [see Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service]. This service will be done at additional costs.
    • If the User conducts their own genotyping, Core Staff can review genotyping results at an additional cost.
  12. If the Core performs genotyping, they will report back to the User the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If the user conducts their own genotyping, the same information will be reported back to the Core.

What to Expect

  1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
    • Design completion or verification
    • Reagent ordering
    • Embryo electroporation
  2. A minimum of 5 live-born offspring. If less than 5 live-born offspring are produced, the electroporation will be repeated for an additional 100 embryos.
  3. At least one live-born animal with the desired genome editing event (one founder animal).
  4. If one founder is not identified:
    • If the guide RNAs were (i) tested in mouse embryos by the Core and the Core approved their use based on the genotyping results and (ii) the Core conducted the founder genotyping, the Core can review results, redesign the approach and reagents if needed, and the electroporation can be repeated. Project costs associated with re-design and additional embryo electroporation will be reduced by 50%. Costs associated with purchase of new guide RNAs and donor DNAs will not be reduced.
    • If the guide RNAs were not tested in mouse embryos or the user conducts their own genotyping, there will be no reduced costs for additional work.
  5. Depending on the desired allele, conventional PCR is generally sufficient to detect evidence of genome editing:
    • Point mutation alleles: Typically, there will be almost no size difference between the edited and unedited allele. However, the inclusion of a novel restriction site in the repair template (introduced by silent mutation) can greatly facilitate detecting targeted animals. Sanger sequencing of PCR products from the desired allele is necessary to verify the sequence at the target site.
    • Epitope tag and conditional KO alleles: The inserted sequence in a correctly targeted allele will create a shift in the size of a PCR product between the edited and unedited allele. Sanger sequencing of PCR products from the desired allele is necessary to confirm the correct sequence at the target site.
    • Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at this stage. Moreover, the various alleles found in a founder can be passed onto the next generation. Thus, when breeding founders, the resulting N1 offspring must be PCR genotyped and Sanger sequenced to assess which alleles were inherited by which animals. A colony should be established from N1 animals harboring the same sequence confirmed HDR allele. We recommend that N1 animals be backcrossed to wild-type animals. Intercrossing N1 animals is not recommended.