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July 22, 2019

microRNA-driven gene editing with RNA machines

The Hirohide Saito lab shows its miRNA switch technology can turn on the CRISPR-Cas9 system to edit genes in living human cells.

Major advances in gene editing suggest the possibility of curing many diseases in the near future. The CRISPR-Cas9 system has dominated the field of gene editing since its discovery due to its relative ease and low cost. One of the biggest challenges preventing it from becoming mainstream medical technology, however, is the specificity and safety with which scientists can use it to modify a gene. Incorrect edits can have greater health repercussions than the disease itself. A new report by the Hirohide Saito laboratory describes RNA technology that significantly enhances accuracy of CRISPR-Cas9 gene editing.

The technology is based on the laboratory's microRNA (miRNA) switch, which turns the expression of a specific gene on or off depending on the presence of specific miRNA in a cell. There are approximately 2600 miRNAs known in the human body. Scientists have realized that the expressions of these miRNAs vary with the cell type, thus making miRNAs a potential intracellular fingerprint.

By responding to different miRNA activity levels, Saito's team has shown how the miRNA switch can identify and purify specific cell types.

"When preparing different cell types from iPS cells, we may get a heterogeneous population. But for cell therapies, we need to eliminate undesired populations. Our miRNA switch can eliminate undesired cells for high purity," explains CiRA Professor Saito.

In the newest report, the researchers show how the miRNA switch is compatible with CRISPR-Cas9 technology. Cas9 is an enzyme that is required for the gene editing.

"AcrllA4 is a Cas9 inhibitor. We designed our miRNA switch to turn on and off the activation of AcrllA4 production to control the gene editing in specific cell types," says Dr. Moe Hirosawa, who first-authored the study.

Hirosawa designed miRNA-AcrllA4 switches and delivered them into a population of different cell types as synthetic mRNAs.

"Our switch turns off AcrllA4 expression in the presence of target miRNAs," says Saito.

Switching off the AcrllA4 expression allows the cell to switch on the CRISPR-Cas9 system, because the Cas9 enzyme is no longer blocked. However, this effect only occurs if the cell expresses the target miRNA.

The same system was proven in the CRISPR-based gene activation system dCas9-VPR, a commonly used derivative of Cas9, in which dCas9-VPR activates a gene rather than edits it.

"Conditional gene editing and gene activation dependent on cellular conditions may reduce side effects, which may be important for future clinical applications." says Saito.

Paper Details
  • Journal: ACS Synthetic Biology
  • Title: Cell-type-specific CRISPR activation with microRNA-responsive AcrllA4 switch
  • Authors: Moe Hirosawa, Yoshihiko Fujita, and Hirohide Saito
  • Author Affiliations:
    1. Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
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