Genome Res:beware! In vitro transcription CRISPR RNA will trigger an immune response

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for many kinds of organisms, CRISPR-mediated genome editing has become a construct of the disease model is a powerful tool, but its clinical application is being developed. Pre-assembled CRISPR-Cas9 ribonucleoprotein（ribonucleoprotein, RNP）complexes by recombinant Cas9 protein and in vitro transcribed guide RNA（gRNA, can be transported to the cells, without the presence of the exogenous DNA integrated into the host genome of the risk, and have fewer off-target effects. However, in a new study, Korean researchers found that in vitro transcription of gRNA in vitro transcribed gRNA, IVT gRNA containing a 5’ triphosphate or 5’ppp）group, it will activate the human cells in the immune response, thereby leading to cell death. Related research results published in 2018 3 month’s Genome Research journal, The paper entitled“CRISPR RNAs trigger innate immune responses in human cells”.

pictures from Sojung Kim.

papers communication writers, from Korea basis Science Institute of Jin-Soo Kim said,“CRISPR-Cas9 RNP is now widely used in biomedical research, and in therapeutic genome editing aspect is promising.” Therefore, the technology is from the bio-medical applications into clinical applications requires a thorough study of the potentially harmful host immune responses.

In this study, these researchers used purified Cas9 and the IVT gRNA complexes formed in transfected human cervical cancer cells（HeLa cell line and mouse embryonic fibroblasts. In these cells, the 5’ppp gRNA trigger Type I interferon-mediated immune response, and interferon activation of antiviral effector proteins, such as DDX58 expression increased, thereby leading to cell death.

In addition, this study confirms that the 5’ppp gRNA of human T cell cytotoxicity. Kim said,“on T cells for genome editing in the development of the treatment of cancer and HIV infection of the cell therapy has great potential. T cells of the gRNA in the 5’ppp Group is very sensitive.” T-cell surface receptor CCR5 involved in HIV viruses, therefore, is the development of resistance to HIV infection therapy is a promising target. Kim and other witnesses utilitarian with a phosphatase-pre-treated IVT gRNA, removing the 5′ – ppp groups, this will significantly reduce the T cells trigger the host immune response and cytotoxicity. After phosphatase treatment of the IVT gRNA will increase the CCR5 mutation efficiency, resulting in this HIV coreceptors on the cell surface of the presenting decreased significantly.

this study elucidated the CRISPR-Cas9 or CRISPR-Cpf1 ribonucleoprotein complexes in the IVT gRNA activation of the immune response of human cells to the cytotoxic effects, and to provide a prevent the host immune response while maintaining a high mutation efficiency of a new method for CRISPR-mediated genome editing in the treatment of the efficient application of the Foundation.

original source:

Sojung Kim, Taeyoung Koo, Hyeon-Gun Jee et al. CRISPR RNAs trigger innate immune responses in human cells. Genome Research, March 2018, 28(3), doi:10.1101/gr. 231936. 117return to Sohu, view more

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