posted on 2021-06-16, 16:51authored bySeunghyeon Shin, Su Hyun Kim, Jae Seong Lee, Gyun Min Lee
A platform,
based on targeted integration of transgenes using recombinase-mediated
cassette exchange (RMCE) coupled with CRISPR/Cas9, is increasingly
being used for the development of mammalian cell lines that produce
therapeutic proteins, because of reduced clonal variation and predictable
transgene expression. However, low efficiency of the RMCE process
has hampered its application in multicopy or multisite integration
of transgenes. To improve RMCE efficiency, nuclear transport of RMCE
components such as site-specific recombinase and donor plasmid was
accelerated by incorporation of nuclear localization signal and DNA
nuclear-targeting sequence, respectively. Consequently, the efficiency
of RMCE in dual-landing pad human embryonic kidney 293 (HEK293) cell
lines harboring identical or orthogonal pairs of recombination sites
at two well-known human safe harbors (AAVS1 and ROSA26 loci), increased
6.7- and 8.1-fold, respectively. This platform with enhanced RMCE
efficiency enabled simultaneous integration of transgenes at the two
sites using a single transfection without performing selection and
enrichment processes. The use of a homotypic dual-landing pad HEK293
cell line capable of incorporating the same transgenes at two sites
resulted in a 2-fold increase in the transgene expression level compared
to a single-landing pad HEK293 cell line. In addition, the use of
a heterotypic dual-landing pad HEK293 cell line, which can incorporate
transgenes for a recombinant protein at one site and an effector transgene
for cell engineering at another site, increased recombinant protein
production. Overall, a streamlined RMCE platform can be a versatile
tool for mammalian cell line development by facilitating multigene
expression at genomic safe harbors.