Abstract
Traditional cell line development is based on random genomic integration
of transgenes. Random integration leads to unpredictable expression and
results in clonal heterogeneity requiring a tedious screening procedure.
Therefore, a new strategy is needed to establish clones that exhibit
stable transgene expression. Here, we performed CRISPR/Cas9-mediated
site-specific integration (SSI) to incorporate a landing pad (LP;
containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and
strong expression. Site-specific integration of LP on Fer1L4 was
demonstrated by sequencing results representing the swapped sequences in
mCherry-expressing cells. We then performed Cre/Lox-based
recombinase-mediated cassette exchange (RMCE) to exchange LP with a
targeting vector (TV; containing GFP) in clones established by
CRISPR/Cas9-mediated SSI. The success of Cre/Lox-based RMCE was
evidenced by sequencing results representing the swapped sequences in
GFP-expressing cells. Furthermore, the swapped clones expressing GFP was
enriched up to 80%, indicating that the efficiency of Cre/Lox-based
RMCE would be sufficient to swap pre-existing cassettes with
gene-of-interest (GOI). Taken together, our study provides a new
platform for Cre/Lox-based RMCE to iteratively establish stable clones
from existing ones previously established by SSI at a genomic hotspot.