Transgene

DNA repair

Plasmid

Cell culture

Biology

Gene

Genome editing

Cas9

Chinese hamster ovary cell

Molecular biology

Homology (biology)

Computational biology

Cell biology

CRISPR

Genetics

DNA mismatch repair

Homology directed repair

Scientific Reports

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway