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

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.