Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing
Hiroshi Kiyonari, Mari Kaneko, Takaya Abe, Aki Shiraishi, Riko Yoshimi, Kenichi Inoue, Yasuhide Furuta
Abstract
However, in vivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.