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Targeted Knockout of the dhfr, glul, bak1, and bax Genes by the Multiplex Genome Editing in CHO Cells

  • BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY
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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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Authors and Affiliations

  1. Skryabin Institute of Bioengineering, Federal Research Center “Fundamentals of Biotechnology”, Russian Foundation for Basic Research, Moscow, Russia

    N. A. Orlova, E. A. Gayamova, M. V. Sinegubova, S. V. Kovnir & I. I. Vorobiev

  2. Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, Moscow, Russia

    L. K. Dayanova & I. I. Vorobiev

Authors
  1. N. A. Orlova
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  2. L. K. Dayanova
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  3. E. A. Gayamova
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  4. M. V. Sinegubova
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  5. S. V. Kovnir
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  6. I. I. Vorobiev
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Corresponding author

Correspondence to N. A. Orlova.

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The authors declare that they have no conflicts of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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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

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