Efficient knock-in at the chicken ovalbumin locus using adenovirus as a CRISPR/Cas9 delivery system

In this study, efficient knock-in (KI) of human epidermal growth factor (hEGF) cDNA at the ovalbumin (OV) locus in cultured chicken cells was achieved using adenovirus as a delivery for CRISPR/Cas9 elements and optimizing donor vector construction. The strategy of recruiting donor DNA to the insertion site further improved the KI efficiency. The inserted hEGF cDNA can expressed in primary oviduct cells and secreted hEGF promoted proliferation of Hela cells. Moreover, we achieved efficient KI in blastoderm cells without altering their induction in vitro and obtained germline chimeric KI chicken embryos by transplanting KI blastoderm cells as well as injecting adenovirus directly, in vivo. Our results provided an efficient KI method for chicken cells and embryos, and lay the foundation for more convenient production of KI chicken at the OV locus, which will promote the development of oviduct-specific bioreactor.