Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

Abstract

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

A novel Bxb1 integrase RMCE system for high fidelity site‐specific integration of mAb expression cassette in CHO Cells

“…Recent work has demonstrated the power of this technology to engineer the genomes of numerous organisms including CHO cells. CRISPR/Cas9 has been used to induce integration of large gene cassettes using homologous recombination as well as microhomology mediated end joining (MMEJ) (Lee et al, 2016;Nakade et al, 2014;Sakuma et al, 2015). While antibiotic selection is often used to enrich for integration of a gene cassette, we opted to include a fluorescent marker in our landing pads allowing enrichment and cloning of cell lines containing the RMCE system by FACS (Lee et al, 2016).…”

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

“…Interestingly, the microhomologies were frequently found in the majority of repaired sites of the DSBs induced by CRISPR/Cas in mice [18] and human cells (more than half of the deletions [19]). Taking advantage of the high frequency of MMEJ, we recently developed the highly efficient and convenient CRISPR/Cas or transcription activator-like effector nuclease (TALEN)-based precise integration into the target chromosome (PITCh) system that harnesses MMEJ to knock-in a gene cassette into target genomic loci with extremely short (≤40 bp) microhomologies in cultured cells [20–22], silkworms [20], frogs [20], and zebrafish [23]. In addition to its high efficiency, the PITCh system has the potential to omit the laborious steps of constructing a targeting vector for gene cassette knock-in by the use of microhomologies.…”

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…Correct tyrosine sulfation appears to not only modulate protein stability (Zhong et al, 2007), but also to influence the biological activity and binding F I G U R E 1 Overview of N-glycosylation in different wild type expression platforms (adapted from Chung, Majewska, Wang, Paul, & Betenbaugh, 2017) affinities by affecting protein-protein interaction (Stone, Chuang, Hou, Shoham, & Zhu, 2009 at any desired location in the genome inducing a double-strandedbreak (DSB), which will be repaired by the cell's own mechanisms (Epinat et al, 2003;Kim, Lee, Kim, Cho, & Kim, 2009;Miller et al, 2011). Inducing the NHEJ mechanism is the preferred way if the aim is to KO a gene and the corresponding function, whereas initiating HDR is the method of choice to introduce new sequences into the host's genome (Sakuma et al, 2015;Santiago et al, 2008). Inducing the NHEJ mechanism is the preferred way if the aim is to KO a gene and the corresponding function, whereas initiating HDR is the method of choice to introduce new sequences into the host's genome (Sakuma et al, 2015;Santiago et al, 2008).…”

“…DSBs are either corrected by the error-prone nonhomologous end joining (NHEJ) pathway resulting in small insertions/deletions (InDels) of nucleotides, thus, most likely in a frame-shift/loss-offunction mutation (Davis and Chen, 2013) or, if a suitable homologymediating template is present, the DNA cut is repaired through the homology-directed repair (HDR) pathway (Liang, Han, Romanienko, & Jasin, 1998). Inducing the NHEJ mechanism is the preferred way if the aim is to KO a gene and the corresponding function, whereas initiating HDR is the method of choice to introduce new sequences into the host's genome (Sakuma et al, 2015;Santiago et al, 2008). In contrast to NHEJ, sequence knockin (KI) via HDR occurs at a much lower frequency in most if not all available expression systems (Heyer, Ehmsen, & Liu, 2010;Miyaoka et al, 2016).…”