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.
Highlights
Designable and controllable knock-in of large gene cassettes has been long desired for animal cell engineering, including production of biopharmaceutical proteins by Chinese hamster ovary (CHO)cells [1]
According to the very recent report, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene knock-in resulted in off-target integration of donor DNA as well as on-target integration in CHO-K1 cells [7]
To obtain an optimal level of exogenous gene expression, we have chosen the hypoxanthine phosphoribosyltransferase 1 (HPRT1) locus, which was previously demonstrated to be an ideal locus for stable expression of exogenous gene cassettes in human fibrosarcoma cells [14]
Summary
Designable and controllable knock-in of large gene cassettes has been long desired for animal cell engineering, including production of biopharmaceutical proteins by Chinese hamster ovary (CHO). NHEJ-based methods reduce the labor of constructing targeting vectors, and overcome the difficulty of gene knock-in in cells and animals with low HR frequency. The PITCh system utilizes microhomology-mediated end-joining (MMEJ) instead of NHEJ or HR, resulting in precise gene knock-in through extremely short microhomologies (≤40 bp). Since these microhomologies can be added by PCR or insertion of synthesized oligonucleotides, the construction of the donor vector for the PITCh system (PITCh vector) does not require PCR amplification of genomic DNA sequence. In CHO cells, programmable nuclease-mediated gene knock-in has mainly been performed by NHEJ-dependent methods, because they have regarded as one of the HR-inactive cells [3,4,7].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
