Avian transgenesis has served as a suitable approach to generate bioreactors for the manufacturing of recombinant proteins. Production in chicken cells comes with significant advantages over other systems including providing the human-like glycosylation on target proteins. In this regard, the oviduct-specific ovalbumin promoter has been one of the ideal candidates to drive the expression of transgenes. Previous plasmid-based studies on the regulatory sequences of the ovalbumin promoter have led researchers to exploit ovalbumin regulatory elements out of their native genomic context (ex situ) to direct transgene expression in the transgenic chicken bioreactors. In this study, we used CRISPR/Cas9 technologies to engineer the genome and epigenome of the ovalbumin promoter and coding sequence in a non-oviduct chicken cell. We show that CRISPR-mediated deletion of some distal ovalbumin promoter sequences (SDRE and NRE elements) can lead to the significant expression of the ovalbumin gene (more than 104-fold compared to that in the wild-type DF1 cell), and also a knocked-in reporter, can function in an estrogen-independent manner. Also, this study is the first record of the CRISPR/Cas9 guided epigenome engineering in chicken cells. We showed that epigenome modulation using CRISPR-mediated transcriptional activation (CRISPRa) can activate the ovalbumin gene in DF-1 cells (non-oviduct cells). In conclusion, through this research, we were able to use the predominant ability of the ovalbumin promoter to produce exogenous proteins via CRISPR- based genome and epigenome engineering in chicken cells.

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