Background:
Conventional methods applied to develop recombinant CHO (rCHO)
cell line as a predominant host for mammalian protein expression are
limited to random integration approaches, which can prolong the process
of getting the desired clones for months. CRISPR/Cas9 could be an
alternative by mediating site-specific integration into
transcriptionally active hot spots, promoting homogenous clones, and
shortening the clonal selection process. However, applying this approach
for the rCHO cell line development depends on an acceptable integration
rate and robust sites for the sustained expression.
Methods and results:
In this study, we aimed at improving the rate of GFP reporter
integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1
genome via two strategies; these include the PCR-based donor
linearization and increasing local concentration of donor in the
vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin
tethering approach. According to the results, compared to the
conventional CRISPR-mediated targeting, donor linearization and
tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in
efficiency; among on-target clones, 84% and 73% were determined to be
single copy by the quantitative PCR, respectively. Finally, to evaluate
the expression level of the targeted integration, the expression
cassette of hrsACE2 as a secretory protein was targeted to the Chr3
pseudo-attP site by applying the established tethering method. The
generated cell pool reached 2-fold productivity, as compared to the
random integration cell line.
Conclusion:
Our study suggested reliable strategies for enhancing the
CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a
potential candidate for the sustained transgene expression, which might
be applied to promote the rCHO cell line development.