Knock-in of donor plasmids in sheep oocytes

KI of donor plasmids was attempted using laser-assisted cytoplasmic injection^{20 (link)} of in vitro matured oocytes after 18 h of maturation and in vitro fertilized embryos 6 hpi with 6 pL of a solution containing 67 ng/μL of in vitro transcribed gRNA, 167 ng/μL of Cas9 protein (PNA Bio) and 133 ng/μL of donor plasmid. Injected MII oocytes were subsequently co-cultured with cumulus-oocyte-complexes (COCs) and in vitro fertilized following procedures previously described for sheep^{26 (link)}. Embryos were scored for developmental stage reached at day 7–8. Embryos that reached blastocyst stage were lysed as described above and underwent whole-genome amplification using the Repli-G Mini kit (Qiagen). Target regions were amplified using primers developed using Primer3 (Supplementary Information, Fig. S1 and Table S4)^{39 (link),40 (link)}. PCR was performed on a thermal cycler with 12.5 μL LongAmp Taq 2X Master Mix (NEB), 9.5 μL of H₂O, 1 μL of each primer at 10 mM and 1 μL of DNA for 5 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at anneal temp (Supplementary Information, Table S4) and 4 min at 65 °C, followed by 15 min at 65 °C. Products were visualized on a 1% agarose gel using a gel imager, purified using the QIAquick Gel Extraction Kit (Qiagen) and Sanger sequenced (GeneWiz).