Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems

Ryusei Iwao,Mai Murakami,Yoshinori Kawabe,Masamichi Kamihira,Akira Ito,M Shimada

doi:10.1051/matecconf/202133307001

Abstract

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

Highlights

Chinese hamster ovary (CHO) cells have been widely used as a host for biopharmaceutical protein production because they can produce proteins with post-translational modifications such as glycosylation and proper folding (Fisher et al, 2015)
High producer cells have been conventionally screened from stable pools with random integration of transgenes, generated using gene amplification methods such as dihydrofolate reductase (DHFR)/methotrexate (MTX) and glutamine synthetase (GS)/methionine sulfoximine (MSX) systems
We evaluated the efficiency of genome editing using T7 endonuclease I (T7EI) assay

Summary

Introduction

Chinese hamster ovary (CHO) cells have been widely used as a host for biopharmaceutical protein production because they can produce proteins with post-translational modifications such as glycosylation and proper folding (Fisher et al, 2015). High producer cells have been conventionally screened from stable pools with random integration of transgenes, generated using gene amplification methods such as dihydrofolate reductase (DHFR)/methotrexate (MTX) and glutamine synthetase (GS)/methionine sulfoximine (MSX) systems. Guided nucleases have been developed for specific modification of target genes on the genome (Kim and Kim, 2014). These methods cause generation of DNA doublestrand breaks (DSBs) at desired sites on the genome, and gene knock-out and knock-in at the target site rely on the DSB repair mechanisms of cells, comprising two major pathways: non-homologous end joining (NHEL) and homologous recombination repair (HDR). HDR-mediated gene modification is frequently used for targeted knock-in of transgenes, the knock-in efficiency is generally known to be low

Methods

Results

Conclusion