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.

中文翻译：

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CRISPR/Cas9技术在小鼠受精卵中的应用获得重复、缺失和倒位影响核型稳定性

摘要

在现代分子和细胞生物学中，CRISPR/Cas9技术已广泛应用于人类和动物基因组的靶向修饰。在这项工作中，我们使用分子细胞遗传学方法分析了 18 个小鼠成纤维细胞系 (MF) 的核型，这些细胞系的基因组中有一个用 CRISPR/ Cas9 编辑的Cntn6基因。Cntn6基因的修饰为2374kb的重复、1137kb的缺失以及类似大小的倒位。此外，还对携带完整Cntn6基因等位基因的小鼠胚胎成纤维细胞 (MEF) 的 5 个对照系进行了细胞遗传学分析。研究表明，MF 品系存在高水平的多倍体，其中Cntn6基因倒位为杂合子，Cntn6 基因倒位为纯合子和杂合子。Cntn6基因重复 (20–46%)，以及 6 号染色体的单体性 (1–9%) 和三体性 (1–8%)。值得注意的是，在携带缺失和重复的 MEF 系中未检测到三体性。化合物中Cntn6基因缺失，多倍体细胞比例极小（1.5-5.7%）。因此，获得的数据表明经过 CRISPR/Cas9 基因组编辑的细胞系的核型不稳定。