The genome of oocytes can be edited in vivo in the zebrafish ovary

Oocytes, the irreplaceable gametes for generating a new organism, are matured in the ovary of living female animals. It is unknown whether any genetic manipulations can be applied to oocytes inside the living ovaries. As a proof-of-concept, we here demonstrate genetic amendments of zebrafish immature oocytes within the ovary. Oocyte microinjection in situ (OMIS) stimulates tissue repair responses, but some of microinjected immature oocytes are matured, ovulated and fertilizable. By OMIS-mediated Cas9 approach, ntla and gata5 loci of oocytes arrested at prophase I of meiosis are successfully edited before fertilization. Through OMIS, high efficiency of biallelic mutations in single or multiple loci using Cas9/gRNAs allows immediate manifestation of mutant phenotypes in F0 embryos and multiple transgenes can co-express the reporters in F0 embryos with patterns similar to germline transgenic embryos. Our approach opens a door to efficient modification of the genome and provides a possibility to repair defective oocytes in situ. Following OMIS with rhodamine B, 10-15 microinjected oocytes (still arrested at prophase I) were isolated from the ovary at 5 hours post-OMIS (hpO) or 14 hpO, while uninjected oocytes were isolated at the same time points from the other ovary of the same female.The RNA-seq were performed in these oocytes.