Transgenic Lines of Human Induced Pluripotent Stem Cells ICGi022-A-6 and ICGi022-A-7 with Doxycycline-Inducible Variants of Programmable Nuclease AsCas12a

Pub Date : 2024-03-07 DOI:10.1134/s1062360423060061

S. V. Pavlova, K. R. Valetdinova, T. B. Malankhanova, D. E. Polivtsev, A. A. Malahova, E. V. Grigor’eva, A. I. Shevchenko, S. M. Zakian, S. P. Medvedev

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Abstract

Genome editing in human pluripotent stem cells using programmable nucleases makes it possible to create models of hereditary pathologies using directed transgenesis, gene knockout, and replacement of individual nucleotides in DNA sequences. Using CRISPR/SpCas9-mediated homologous recombination at the AAVS1 locus, clones of human induced pluripotent stem cells (iPSCs) ICGi022-A (Malakhova et al., 2020) were obtained, which carry transgenes of two variants of the nuclease AsCas12a (also known as AsCpf1), recognizing different PAM consensuses, and the reverse doxycycline transgene-dependent transactivator M2rtTA. For each AsCas12a variant, the lines ICGi022-A-6 (AsCas12a, PAM 5'-TTTV-3') and ICGi022-A-7 (AsCas12a, PAM 5'-TYCV-3') were obtained. Using Western blot analysis, it was shown that the addition of doxycycline to the culture medium causes activation of the expression of AsCas12a(TTTV) and AsCas12a(TYCV) proteins. The resulting transgenic iPSC clones were subjected to molecular and cytogenetic analysis. Using quantitative PCR and immunocytochemical analysis, it was shown that they have a high level of mRNA expression of gene markers of pluripotent cells, namely OCT4, NANOG, and SOX2, as well as specific expression of protein markers OCT4, SOX2, SSEA-4, and TRA-1-60. In addition, using iPSCs spontaneous differentiation into embryoid bodies, it was found that transgenic clones can give derivatives of all three primitive germ layers: ectoderm, mesoderm, and endoderm. Cytogenetic analysis showed that transgenic iPSC clones have a normal karyotype, 46,XX.

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用强力霉素诱导的可编程核酸酶 AsCas12a 变异株转基因人诱导多能干细胞 ICGi022-A-6 和 ICGi022-A-7

摘要利用可编程核酸酶在人类多能干细胞中进行基因组编辑，可以通过定向转基因、基因敲除和替换DNA序列中的单个核苷酸来创建遗传病模型。利用 CRISPR/SpCas9 介导的 AAVS1 基因座同源重组，获得了人类诱导多能干细胞（iPSCs）ICGi022-A 的克隆（Malakhova 等人，2020 年），其中携带了两种核酸酶 AsCas12a（又称 AsCpf1）变体的转基因，识别不同的 PAM 共识子和反向多西环素转基因依赖性转录因子 M2rtTA。对于每种 AsCas12a 变体，都得到了品系 ICGi022-A-6（AsCas12a，PAM 5'-TTV-3'）和 ICGi022-A-7（AsCas12a，PAM 5'-TYCV-3'）。通过 Western 印迹分析表明，在培养基中加入强力霉素可激活 AsCas12a(TTTV) 和 AsCas12a(TYCV) 蛋白的表达。对得到的转基因 iPSC 克隆进行了分子和细胞遗传学分析。通过定量 PCR 和免疫细胞化学分析，结果表明它们具有高水平的多能细胞基因标志物 mRNA 表达，即 OCT4、NANOG 和 SOX2，以及蛋白质标志物 OCT4、SOX2、SSEA-4 和 TRA-1-60 的特异性表达。此外，利用自发分化成胚状体的 iPSCs 发现，转基因克隆可产生所有三个原始胚层（外胚层、中胚层和内胚层）的衍生物。细胞遗传学分析表明，转基因 iPSC 克隆的核型正常，为 46,XX。

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