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The Application of CRISPR/Cas9 Technology in Mice Zygotes to Obtain Duplications, Deletions and Inversions Affects Karyotype Stability

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Abstract

In modern molecular and cellular biology, CRISPR/Cas9 technology has been widely used for targeted modification of human and animal genomes. In this work, using molecular-cytogenetics methods, we have analyzed the karyotype in 18 mouse fibroblast cell lines (MFs), whose genome had a Cntn6 gene edited with CRISPR/Cas9. The modifications of Cntn6 gene were 2374-kb duplications, 1137-kb deletions, and inversions of similar size. In addition, cytogenetic analysis was performed for five control lines of mouse embryonic fibroblasts (MEFs) carrying an intact Cntn6 gene allele. The study showed the presence of a high level of polyploidy for MF lines that were heterozygous for Cntn6 gene inversions and homozygous and heterozygous for Cntn6 gene duplications (20–46%), as well as monosomy (1–9%) and trisomy (1–8%) for chromosome 6. It is important to note that no trisomy was detected in MEF lines carrying a deletion and duplication of Cntn6 gene in the compound, and the proportion of polyploid cells was minimal (1.5–5.7%). Thus, the data obtained indicate the destabilization of the karyotype of cell lines that underwent genome editing by CRISPR/Cas9.

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Funding

The authors declare that they have no conflicts of interest. This work was supported by the Russian Foundation for Basic Research (project no. 20-04-00463А) and budget project no.FWNR-2022-0019.

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Authors and Affiliations

  1. Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 630090, Novosibirsk, Russia

    Yu. M. Minina, T. V. Karamysheva & O. L. Serov

  2. Moscow Institute of Physics and Technology, 141701, Dolgoprudny, Moscow oblast, Russia

    A. B. Soroka

  3. Institute of Molecular and Cellular Biology, Siberian Branch, Russian Academy of Sciences, 630090, Novosibirsk, Russia

    N. A. Serdyukova

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  1. Yu. M. Minina
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Correspondence to Yu. M. Minina.

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Statement on the welfare of animals. All procedures were carried out in accordance with an EU Council Regulation (November 24, 1966, 86/609/EEC) and approved by the Bioethics Commission of the Institute of Cytology and Genetics (authorization no. 24 of October 24, 2014).

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Translated by I. Fridlyanskaya

Abbreviations: BSA—bovine serum albumin; CRISPR/Cas9—clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9; FISH—fluorescent in situ hybridization; PCR—polymerase chain reaction; MEF—mouse embryonic fibroblast; MF—mouse fibroblast.

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Minina, Y.M., Soroka, A.B., Karamysheva, T.V. et al. The Application of CRISPR/Cas9 Technology in Mice Zygotes to Obtain Duplications, Deletions and Inversions Affects Karyotype Stability. Cell Tiss. Biol. 17, 557–564 (2023). https://doi.org/10.1134/S1990519X23050085

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