Applying the CRISPR-Cas9 system to targeted transgene integration in the development of recombinant lepidopteran insect cells

Abstract

The clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system is a widely used genome editing tool, but its application for the development of stably transformed lepidopteran insect cells for recombinant protein production has been limited. In the present study, we applied the CRISPR-Cas9 system to the targeted transgene knock-in to efficiently develop recombinant insect cells. A double-strand break (DSB) in a target site of the genome of Trichoplusia ni BTI-TN-5B1-4 (High Five) cells was detected by T7 endonuclease assay after the cells were transfected with a plasmid for genome editing by employing a U6-4 promoter derived from T. ni to drive guide RNA (gRNA). Genomic DNA analysis via PCR confirmed the integration of a transgene into a target site of the High Five cell genome. The length of the homology arm affected the genome editing efficiency, and the use of a 500-bp version proved to be optimal. Via the co-transfection of High Five cells with gRNA-Cas9 plasmid and a donor vector harboring a reporter gene and the neomycin resistance gene, 21 clones of stably transformed cells were established. In 19 of those clones a site-specific knock-in of the reporter gene at the target site was confirmed, which indicates that an exogenous gene can be efficiently integrated into a target site of the High Five cell genome via the CRISPR-Cas9 system, though transcriptional hot spots in the genome should be identified to establish highly productive recombinant cells.