CRISPR-assisted ROSA26 Targeting with Double-stranded DNA (dsDNA) in Mouse Zygotes

CRISPR/Cas-initiated HR in mouse embryos will be used to generate founder animals harboring inserted sequence targeted to the ROSA26 locus. The targeting will be done in embryos using microinjection to introduce the CRISPR reagents. The investigator will provide their own targeting donor developed from one of the vectors available from the Kühn Lab.Vectors available can be found on Addgene:

• www.addgene.org/Ralf_Kuehn/
• Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.BMC Biotechnol. 2016 Jan 16;16(1):4. doi: 10.1186/s12896-016-0234-4.

Refer to Fig. 7 for the constructs that can be used.

The individual investigators will do all donor design and production. For additional resources to assist in donor production, contact the core. It is recommended that the investigator request this service once they have a suitable donor DNA ready for microinjection.

What Will Happen

1. The investigator will initiate a CRISPR-KI request in iLab. The investigator will list the gene name as Rosa26; specify that the investigator created the design; and select from a drop down list of allele types “Rosa26 locus KI”. 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 investigator will be held to verify the standard components described by Chu et al (see reference above) are to be used.
3. The core will provide the published guideRNA. Lab produced guide RNAs will not be used.
4. The investigator will provide 6-8 ug of donor DNA for phenol: chloro form extraction by the core, prior to assembling the microinjection mix.
5. The core will assemble an appropriate microinjection mix for pronuclear microinjections: the guide RNA and Cas9 protein will be complexed into RNPs and the repair template added subsequently, in nuclease-free buffer.
6. 200 C57BL/6J, C57BL/6N, or FVB/NJ embryos collected from superovulated females will be electroporated and transferred to pseudopregnant females. Contact the GERM core to discuss the necessary steps if other strains are desired.
7. Live-born founder animals will be held by the GERM core until 14 days of age and subsequently transferred to the investigator.
8. Genotyping:
   • It is highly recommended that the core perform genotyping of live-born offspring for the correct targeting at the ROSA26 locus[see Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service].The core has optimized long PCR strategies to verify correct targeting at each homology arm.
   • If the investigator conducts their own genotyping, core Staff can review genotyping results at an additional cost.
9. 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 verification
   • 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. The core will not guarantee that at least one live-born animal with targeting at the Rosa26 locus is obtained. However, if genotyping is performed by the core, and no successfully targeted founders are identified, the microinjection will be repeated for another 100 embryos.
4. If the investigator conducts their own genotyping, the core will only repeat the microinjection at 100% cost.
5. For targeting at the Rosa26 locus, a long-range PCR reaction is needed to verify the correct insertion of the homology arms. Additionally, conventional PCR can be used to detect evidence of genome editing around the target site, and to screen for the KI sequence, however neither of these reactions will provide confirmation of a correctly targeted allele. The homology arm PCR products will only be produced in animals with a correctly targeted 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 to assess which animals inherited which alleles, and Sanger sequenced to confirm the targeted allele. 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.