CRISPR-Cas9 Mouse Transgenesis

Mouse transgenesis was performed as previously described [24 (link)]. In brief, embryos for microinjection were collected from the oviducts of superovulated donor females (100% C57BL6/NRj background, Janvier Labs (SE-ZYG-CNP)). Microinjections were carried out with the help of an Axio Observer.D1 microscope (Zeiss) and microinjector devices CellTram and FemtoJet with TransferMan NK2 micromanipulators (Eppendorf). A premixed solution containing the sgRNA (50 ng/µl), Cas9 mRNA (50 ng/µl; TriLink Biotechnologies), Cas9 protein (30 ng/µl; PNA Bio Inc) and the ssODNs(100 ng/µl; IDT) was injected into the male pronucleus with injection capillaries (BioMedical Instruments, BM 100F-10; type PI-1.6) [25 (link)]. One day after the microinjection, 2-cell stage embryos were transferred into the oviducts of pseudo-pregnant foster females.