22 January 2018

One gene is enough

CRISPR activation of one gene turned "adult" cells back into stem cells

Daria Spasskaya, N+1

The researchers were able to obtain induced pluripotent stem cells from differentiated connective tissue cells by injecting them with an artificial transcription activator based on the Cas9 protein. To turn "adult" cells into stem cells, it was enough to activate a single "Yamanaki factor" – Sox2 or Oct4. The study is published in the journal Cell Stem Cell.

Pluripotent stem cells are in great demand in medicine – many different types of cells can be obtained from them, for example, neurons, cardiomyocytes or retinal cells, which can later be used for transplantation into a diseased organ. In addition, stem cells themselves are also used for transplantation – for example, we told how transplantation of induced stem cells cured mice of glaucoma, and injection of stem cells into the brains of people affected by stroke significantly improved the condition of patients.

In 2012, the Nobel Prize in Physiology or Medicine was awarded to Japanese scientist Shinya Yamanaka for developing a technology for converting differentiated cells back into stem cells – the so-called induced pluripotent stem cells (iPSC). It turned out that for this purpose it is necessary to express four proteins in cells – transcription factors Oct4, Sox2, Klf4 and c-Myc, which are conventionally called "Yamanaki factors".

Researchers from the Gladstone Institutes in the USA and Tsinghua University in China used a CRISPR activation system based on a modified inactive Cas9 protein (dCas9) to artificially increase the expression of these factors. In this system, dCas9 is not used for editing, since it lacks nuclease activity, but as a "means of delivering" activator or repressor proteins to a specific part of the genome to control gene expression.

In their work, the scientists used as an artificial activator the dCas9-SunTag-VP64 design developed several years earlier, which ensures the attraction of multiple VP64 activator domains to the regulatory site of the gene at once and thus provides a high level of expression of the selected gene.


The scheme of operation of the artificial activator dCas9-SunTag-VP64. The protein is attracted to the promoter of the desired gene using guide RNA (sgRNA), and activates the transcription of the gene

At first, the researchers used a design for simultaneous activation of two "Yamanaki factors" and three other "stem" factors in mouse embryonic fibroblasts (differentiated connective tissue cells), and made sure that the cells undergo genome remodeling and activation of genes characteristic of stem cells. Markers of the pluripotent state of cells were increased expression of a panel of genes, including Oct4, Sox2, Nanog, Esrrb, Nr5a2 and Utf1.

Next, the researchers began to activate the factors one by one, and found that a high level of activation of a single Sox2 gene, which was achieved by attracting dCas9 to one of its regulatory sites (promoter S-17), provides induction into pluripotent stem cells of both mouse embryonic fibroblasts and fibroblasts isolated from the skin of an adult mouse. The stem cells obtained in this way retained their properties for at least 20 transplants. Activation of the Oct4 factor also led to the transformation of mouse fibroblasts into stem cells. To do this, it turned out to be necessary to direct dCas9 not only to the gene promoter, but also to a remote regulatory site (enhancer).

Despite the fact that CRISPR activation has already been used to induce the expression of Sox2 and Oct4 genes, it has not yet been possible to obtain induced pluripotent stem cells using this approach.

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