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

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.

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems

“…Here we conduct an alternative approach to generate an in vitro orthogonal translation system by utilizing CHO cells stably transfected with eAzFRS. Several studies have demonstrated the use of CRISPR/Cas technology to overexpress a diverse repertoire of proteins from defined genomic loci in CHO cells ( Lee et al, 2015 ; Eisenhut et al, 2018 ; Iwao et al, 2021 ). The genomic loci HPRT1 and C12orf35 were investigated for the generation of stable CHO cell lines using CRISPR/Cas, as reported in a recent study ( Zhao et al, 2018 ).…”

Targeting DNA repair pathways with B02 and Nocodazole small molecules to improve CRIS-PITCh mediated cassette integration in CHO-K1 cells

“…The CRIS-PITCh is known as the MMEJ-interceded integration system to target predefined genomic sites via extremely short homology arms (20-40 bp) which can be easily prepared by PCR or synthetic oligonucleotides 33,34 .…”

CRISPR‐interceded CHO cell line development approaches

“…The CRIS‐PITCh system harnesses MMEJ instead of HR or NHEJ for transgene KI through extremely short microhomology arms (20–40 bp) (Figure 2b) (Kawabe et al, 2018). This short microhomology may be readily introduced to the donor vector by PCR or synthetic oligonucleotide insertion (Iwao et al, 2021). The PITCh system is one of the most prevalent KI systems due to its capacity to integrate large gene cassettes in a backbone‐free, direction‐oriented, and non‐mutagenic manner in a variety of cells and organisms, as well as the ease with which the unique donor vector can be constructed (Sakuma, 2015).…”