AsianScientist (Mar. 5, 2018) – A research group in South Korea has discovered that a molecular tag on synthetic guide RNA (gRNA) used by the CRISPR-Cas9 gene editing machinery triggers an immune response in human cells, leading to cell death. Their study, published in Genome Research, also suggested a method of reducing the immunogenicity of gRNA.
CRISPR-mediated genome editing has become a powerful tool for modeling of disease in various organisms and is being developed for clinical applications. Preassembled Cas9 ribonucleoproteins composed of the recombinant Cas9 protein and in vitro transcribed (IVT) gRNA complexes can be delivered into cells without the risk of foreign DNA integration into the host genome and with fewer off-target effects. These IVT gRNAs typically contain a 5′ triphosphate (5’ppp) moiety.
In this study, a team of researchers at the Institute of Basic Science in South Korea demonstrated that the 5’ppp moiety activates the immune response in human cells, leading to cell death. They first generated and transfected IVT gRNAs complexed with purified Cas9 into human cervical cancer cells (HeLa) and mouse embryonic fibroblasts. Within these cells, 5’ppp gRNAs elicited a type I interferon-mediated immune response. The IVT gRNAs also increased the expression of interferon-activated antiviral effector proteins, such as DDX58, causing approximately 80 percent of the cells to die.
The researchers further demonstrated that the 5’ppp moiety can be removed by a phosphatase in vitro. This leaves the gRNA with a 5′-hydroxyl group instead, which complexes with Cas9 without inducing a strong immune response.
When the researchers applied their method to primary human CD4+ T-cells, a type of immune cell, they were able to achieve targeted mutagenesis at a frequency of 95 percent. They noted that these results are well aligned with earlier research demonstrating that chemically synthesized gRNAs with a 5′-hydroxyl group are much more efficient than IVT gRNAs in human and other mammalian cells.
These findings represent a step forward in the effective therapeutic application of CRISPR-mediated genome editing, said the researchers.
The article can be found at: Kim et al. (2018) CRISPR RNAs Trigger Innate Immune Responses in Human Cells.
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Source: Institute for Basic Science; Photo: Shutterstock.
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