Abstract
The Chinese hamster ovary cell line CHO is widely used for biopharmaceutical production. Genome editing makes it possible to improve the growth properties of cells, their auxotrophy, and the functioning of the apoptosis and autophagy induction systems. Simultaneous editing of multiple genes makes it possible to obtain a cell line with the required genotype faster than several consecutive rounds of genomic knockout, but the probability of success is lower. Simultaneous editing of the dhfr, glul, bak1, and bax genes in the CHO S cells genome yielded 24 clones with signs of auxotrophy for thymidine and glutamine. Five of them turned out to be dhfr+/–, all five contained a knockout of one or two glul alleles. In one clone, 7 out of 8 target alleles were inactivated by a frameshift, and the second dhfr allele was partially inactivated by insertion of the GAA triplet, which reduced the enzyme activity 2.5 times. The probability of simultaneous knockout of both dhfr alleles increased to 50% when the genome was edited with a pair of guide RNAs directed to one exon of the dhfr gene.
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REFERENCES
Cao, Y., Kimura, S., Park, J.Y., et al., Chromosome identification and its application in Chinese hamster ovary cells, BMC Proc., 2011, vol. 5, suppl. 8, art. O8.
Derouazi, M., Martinet, D., Besuchet Schmutz, N., et al., Genetic characterization of CHO production host DG44 and derivative recombinant cell lines, Biochem. Biophys. Res. Commun., 2006, vol. 340, no. 4, pp. 1069–1077.
Orlova, N.A., Kovnir, S.V., and Khodak, Y.A., et al. high-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors, PLoS One, 2019, vol. 14, no. 7, art. e0219434.
Santiago, Y., Chan, E., and Liu, P.Q., et al., Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases, Proc. Natl. Acad. Sci. U. S. A., 2008, vol. 105, no. 15, pp. 5809–5814.
Fan, L., Kadura, I., Krebs, L.E., et al., Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells, Biotechnol. Bioeng., 2012, vol. 109, no. 4, pp. 1007–1015.
Kovnir, S.V., Orlova, N.A., Khodak, Yu.A., et al., Approaches to controlled co-amplification of genes for production of biopharmaceuticals: study of the insertion and amplification dynamics of genetic cassettes in the genome of Chinese hamster ovary cells during co-expression of compatible pair of plasmids, Bull. Exp. Biol. Med., 2017, vol. 163, no. 2, pp. 245–249.
Heffner, K.M., Wang, Q., and Hizal, D.B., et al., Glycoengineering of mammalian expression systems on a cellular level, Adv. Biochem. Eng. Biotechnol., 2021, vol. 175, pp. 37–69.
Lee, J.S., Ha, T.K., Park, J.H., and Lee, G.M., Anti-cell death engineering of CHO cells: co-overexpression of Bcl-2 for apoptosis inhibition, Beclin-1 for autophagy induction, Biotechnol. Bioeng., 2013, vol. 110, no. 8, pp. 2195–2207.
Macaraeg, N.F., Reilly, D.E., and Wong, A.W., Use of an anti-apoptotic CHO cell line for transient gene expression, Biotechnol. Progr., 2013, vol. 29, no. 4, pp. 1050–1058.
Grav, L.M., Lee, J.S., Gerling, S., et al., One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment, Biotechnol. J., 2015, vol. 10, no. 9, pp. 1446–1456.
Ronda, C., Pedersen, L.E., and Hansen, H.G, et al., Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool, Biotechnol. Bioeng., 2014, vol. 111, no. 8, pp. 1604–1616.
Tarasevich, A., Filatov, A., Pichugin, A., and Mazu-rov, D., Monoclonal antibody profiling of cell surface proteins associated with the viral biofilms on HTLV-1 transformed cells, Acta Virol., 2015, vol. 59, no. 3, pp. 247–256.
Sentmanat, M.F., Peters, S.T., Florian, C.P., et al., A survey of validation strategies for CRISPR-Cas9 editing, Sci. Rep., 2018, vol. 8, no. 1, p. 888.
Brinkman, E.K., Chen, T., Amendola, M., and van Steensel, B., Easy quantitative assessment of genome editing by sequence trace decomposition, Nucleic Acids Res., 2014, vol. 42, no. 22, art. e168.
Hsiau, T., Maures, T., Waite, K., et al., Inference of CRISPR edits from Sanger trace data, bioRxiv, 2018, art. 251082.
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Orlova, N.A., Dayanova, L.K., Gayamova, E.A. et al. Targeted Knockout of the dhfr, glul, bak1, and bax Genes by the Multiplex Genome Editing in CHO Cells. Dokl Biochem Biophys 502, 40–44 (2022). https://doi.org/10.1134/S1607672922010082
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DOI: https://doi.org/10.1134/S1607672922010082