Generation of Knock-in Mice Using Long Single-Stranded DNA (lssDNA) or Double-Stranded DNA (dsDNA)

CRISPR/Cas-initiated HDR in mouse embryos will be used to generate founder animals harboring inserted sequence (loxP sites, fluorescent reporters, or other complex alleles) 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 microinjection to introduce the CRISPR reagents.

What Will Happen

1. The investigator will initiate a CRISPR-KI request in iLab. The request form requires the gene name and a brief allele description. An IACUC protocol number, mouse transfer information, and a charge account must be provided at this time. CRISPR projects can only be paid for using a BCM-associated account.
2. A consultation meeting between the core and the 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:
     • Conditional alleles: The core will select a critical exon, select guide RNAs, design (a) suitable repair template(s) to insert both LoxP sequences, and design a PCR-based genotyping scheme. The design will be reviewed with the investigator and modifications made if necessary.
     • Reporter and Recombinase KI alleles: With information from the investigator describing the location of the desired KI, the core will select guide RNAs, design a suitable repair template to insert the desired sequence, and design a PCR-based genotyping scheme. The design will be reviewed with the Investigator and modifications made if necessary.
     • Complex alleles: With information from the investigator describing the desired allele, the core will select guide RNAs, design a suitable repair template to insert the specified sequence, and design a PCR-based genotyping scheme. The core has the right to decline producing a design for any overly complicated allele that may be difficult to target in mouse zygotes using CRISPR reagents. Furthermore, the core may suggest that certain complex projects be attempted in ES cells through traditional targeting.
   • Review genome editing approaches designed by the 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. Investigators can opt to have the guide RNAs tested for genome editing efficiency in mouse zygotes prior to the production attempt [view Guide RNA testing in mouse zygotes]. This service is strongly recommended for precise genome edits requiring costly repair donor templates.
7. The core will provide a quote for the synthesis of the repair template, so that the investigator may have a PO generated for the purchase of the donor DNA.
8. Depending on the location of the microinjection, the core will assemble an appropriate microinjection mix:
   • For cytoplasmic injections the mix will contain the guide RNA(s), DNA repair template(s), and Cas9 mRNA in nuclease-free buffer at concentrations determined by the core.
   • For pronuclear microinjections (projects with dsDNA repair templates) the guide RNA(s) and Cas9 protein will be complexed into RNPs and the microinjection mix will be prepared with the repair template 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 microinjected and transferred to pseudopregnant females. Please contact the GERM core to discuss the necessary steps if other strains are desired.
10. Live-born founder animals will be held by the GERM core until 14 days of age and subsequently transferred to the investigator.
11. Genotyping:
    • It is recommended that the core perform genotyping of live-born offspring for the desired genome editing event [view Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service]. This service will be done at an additional cost.
    • If the investigator 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 investigator 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 investigator 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 microinjection
2. A minimum of 5 offspring transferred to the investigator’s colony. If less than 5 offspring are transferred, the microinjection will be repeated for an additional 100 embryos with a new preparation of the donor DNA.
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 will review the results to troubleshoot any potential limitations. A repeat microinjection may be the best approach for obtaining a founder. If a redesign is needed, project costs associated with re-design and additional embryo microinjections will be reduced by 50%. For reagent purchases on project redesigns the core will cover the cost of new gRNA(s), but the investigator will be responsible for the cost associated with purchase of the new donor DNA(s).
   • If the guide RNAs were not tested in mouse embryos or the investigator 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. Genotyping schemes will be designed to only amplify PCR products in the correctly targeted allele. Additionally, 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.
6. 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.