Construction of a gene knockout CHO cell line using a simple gene targeting method
© Aga et al. 2015
Published: 14 December 2015
Therapeutic antibodies have become an important focus of the biopharmaceutical industry. The Chinese hamster ovary (CHO) cell line is a major host for therapeutic antibody production. To construct productive CHO cell lines, two major transfection methods are commonly used, i.e., random integration and gene targeting. Random integration is a common method in which randomly integrated transgenes cause variation in antibody productivity because they are located in various chromosomal regions that affect transgene expression levels. Recently, gene-targeting methods, in which exogenous genes are inserted into a specific chromosomal region, have improved remarkably. Gene targeting is based on homologous recombination using sequences targeting a specific genomic region of the host cell. Homologous sequences located on both sides of the exogenous gene are used. We used the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system as a gene-targeting method. The CRISPR-Cas9 system induces double-strand breaks (DSBs) via guide RNA and Cas9, which increases the efficiency of homologous recombination . Guide RNA hybridizes to a target integration site and induces Cas9 protein expression, leading to DSB. Finally, the Cas9 protein cuts genomic DNA. In this study, we constructed a simple gene-targeting method in CHO cells using the CRISPR-Cas9 system in which CRISPR vectors induce DSBs and gene-targeting vectors are inserted at the DSB site. In the conventional method, gene-targeting vectors should contain homology arms for effective recombination. In this study, we used the CRISPER system without homology arms for gene-targeted recombination.
Materials and methods
We constructed a CRISPR-Cas9 vector that expresses a guide RNA sequence targeting a region on chromosome O. Chromosome O was selected based on a previous classification of gene-amplified CHO cell chromosomes in order of decreasing size and assigned letters from A to T by fluorescence in situ hybridization (FISH) . The CRISPR targeting sequence was determined from the BAC clone Cg0031N14, which contained the chromosome O sequence. Gene targeting vectors (pcDNA-GFP-DHFR) with or without target site homology arms were constructed from BAC clone Cg0031N14. The percentage of exogenous gene integration into chromosome O was determined by a FISH analysis. Total RNA extracted from E14Tg2a (mouse ES cells) was kindly provided by Dr. Tohru Kimura, Kitasato University, Kanagawa, Japan.
Using the CRISPR-Cas9 system without homologous regions, we performed knockouts of de novo DNA methyltransferase genes in CHO cells. The cellular productivity of a gene-of-interest (GOI) is known to decrease during long-term cultivation. DNA methylation is closely related to this decrease in productivity. We constructed methyltransferase-knockout CHO cells for stable production. The expression levels of the de novo DNA methyltransferases Dnmt3a, 3b, and 3L in CHO cells have not been examined previously. We investigated the expression levels of these methyltransferases using RT-PCR. E14Tg2a cells (mouse ES cells) were used as a positive control and express Dnmt3a, 3b, and 3L. Only Dnmt3a was expressed in CHO-K1 cells, while Dnmt3b and 3L were not detected. Therefore, we focused on the downregulation of Dnmt3a expression in CHO cells using the CRISPR-Cas9 system without homologous regions that we developed. The CRISPR-targeting sequence was determined based on the Dnmt3a activation site in exon 19 and Dnmt3a expression was knocked out (Target 1). The CRISPR vector was constructed 5 bases from the Dnmt3a stop codon as the second target (Target 2). For Targets 1 and 2, Dnmt3a knock-out CHO cell lines were constructed using the CRISPR-Cas9 system without homologous regions.
We were able to efficiently insert exogenous genes into a specific genomic region using the simple CRISPR-Cas9 vector system; additionally, Dnmt3a knock-out CHO cell lines were successfully constructed using this method.
This work was partly funded by a grant for the Project focused on developing key technology of discovering and manufacturing drug for next-generation treatment and diagnosis from the Ministry of Economy, Trade and Industry of Japan and partly by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (No.26630433, 26249125). We are grateful to Dr. Tohru Kimura for providing total RNA extracted from E14Tg2a.
- Le Cong, F Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick Hsu, Xuebing Wu, Wenyan Jiang, Luciano Marraffini, Feng Zhang: Multiplex genome engineering using CRISPR/Cas systems. Science. 2013, 339 (6121): 819-23.View ArticleGoogle Scholar
- Takeshi Omasa, Yihua Cao, Joon Young Park, Yasuhiro Takagi, Shuichi Kimura, Hidenori Yano, Kohsuke Honda, Shuichi Asakawa, Nobuyoshi Shimizu, Hisao Ohtake: Bacterial artificial chromosome library for genome-wide analysis of Chinese hamster ovary cells. Biotechnol Bioeng. 2009, 104 (5): 986-994.View ArticleGoogle Scholar
- Tomohiro Yoshikawa, Fumi Nakanishi, Seima Itami, Daisuke Kameoka, Takeshi Omasa, Yoshio Katakura, Michimasa Kishimoto, Ken-ichi Suga: Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes. Cytotechnology. 2000, 33 (1-3): 37-46.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.