Efficient generation of non‑mosaic genetically modified marmosets via CRISPR/Cas9 delivery into unfertilized MII oocytes

The common marmoset ( Callithrix jacchus ) is a small nonhuman primate (NHP) with a relatively high reproductive capacity due to its short gestation period and early sexual maturation, making it well suited for producing genetically modified primates. Unlike rodents, marmosets take several years to produce second generation offspring; therefore, analyzing founder-generation animals is especially desirable. However, the frequent occurrence of mosaicism is a major obstacle to this. Our BrdU analyses revealed that DNA replication in marmoset zygotes starts around 8 h after fertilization. We reasoned that the high frequency of mosaicism observed with conventional methods results from genome editing in embryos after or during DNA replication. To induce gene modification before DNA replication, a complex of Cas9 and three or four guide RNAs (gRNAs) targeting the same exon was injected into metaphase II (MII) oocytes just before in vitro fertilization (IVF). Using this approach, we generated genetically modified marmosets targeting three genes. All resulting genetically modified animals were likely non-mosaic knockouts. Our MII oocyte injection approach can be used in combination with any oocyte retrieval (ovary or oviduct) and transfer (oviduct and uterus) conditions. Thus, injection of efficient gRNAs in advance of DNA replication enables efficient generation of non-mosaic knockout animals.