Generation of Transgenic Rat Embryonic Stem Cells Using the CRISPR/Cpf1 System for Inducible Gene Knockout

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Vladimir V. Sherstyuk^1,a* and Suren M. Zakian¹

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

^* To whom correspondence should be addressed.

Received March 23, 2021; Revised May 9, 2021; Accepted May 10, 2021

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Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism. To study functions of the essential genes, as well as the processes of cell differentiation, the method of induced knockout is widely used. The CreERT2/loxP system allows obtaining an inducible knockout in cells expressing tamoxifen-inducible Cre recombinase (CreERT2) and containing loxP sites flanking the target gene by adding 4-hydroxy tamoxifen to the culture medium. However, the rat ESC lines expressing CreERT2 are absent. In this work, we tested three CRISPR/Cas systems for introduction of double-strand breaks into the Rosa26 locus in the rat ESCs and inserted tamoxifen-dependent Cre recombinase into this locus using the CRISPR/Cpf1 system. It was shown that the obtained transgenic rat ESC lines retained the characteristics of pluripotent cells. Tamoxifen-inducible Cre recombinase activity was analyzed using a reporter vector.

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KEY WORDS: embryonic stem cells, rat, CRISPR/Cas, genome editing, Cre recombinase

DOI: 10.1134/S0006297921070051

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