CRISPR/CAS-mediated deletion of the upstream regulatory sequences
enhances the estrogen-independent expression of chicken ovalbumin
Abstract
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 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. Although the inherent limitations on
the ex situ use of ovalbumin promoter have promoted the use of native
ovalbumin promoter for the expression of a transgene, generation of
transgenic chicken is relatively difficult, inefficient, and
time-consuming. To overcome these obstacles, in this study we show that
CRISPR-mediated deletion of some distal ovalbumin promoter sequences in
a non-oviduct cell can lead to the significant expression of the
ovalbumin gene, and also a knocked-in reporter, in an
estrogen-independent manner. These findings overcome the limitation of
cloned promoters, where the promoter regulatory sequences have to be
taken out of their cis context and also their native spatial nuclear
organization into a plasmid.