February 22, 2018 - 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 (RNPs) composed of the recombinant Cas9 protein and in vitrotranscribed (IVT) guide RNA complexes can be delivered into cells without risk of foreign DNA integration into the host genome and with fewer off-target effects. However, in a study published today in Genome Research, scientists discovered in vitro-transcribed gRNAs, containing a 5' triphosphate (5'ppp) moiety, activate the immune response in human cells leading to cell death.
"CRISPR-Cas9 RNPs are now broadly used in biomedical research and hold promise in therapeutic genome editing" says corresponding author Jin-Soo Kim from the Institute for Basic Science (Seoul, South Korea). Thus, translation of this technology from biomedical applications to clinical applications requires a thorough investigation into potentially deleterious host responses.
In this study, researchers generated and transfected IVT gRNAs complexed with purified Cas9, into human cervical cancer cells (HeLa) and mouse embryonic fibroblasts. In these cells, 5'ppp gRNAs elicited a type I interferon-mediated immune response, as well as increased expression of interferon-activated antiviral effector proteins, such as DDX58, resulting in cell death.
Additionally, this study demonstrates the cytotoxic effects of 5'ppp gRNA on human T cells. "Genome editing in T cells holds great promise in cell therapy for the treatment of cancer and HIV infection. T cells were extremely sensitive to the 5' triphosphate group in gRNAs" says Kim. The T cell surface receptor, CCR5, is involved in HIV virus entry and is therefore a promising target for anti-HIV therapies. Kim et al. demonstrated that pretreatment of IVT gRNAs with phosphatase, removes the 5'ppp and significantly reduces T cell host immune response and cytotoxicity. Phosphatase treated IVT gRNAs displayed increased mutation efficiency against CCR5 resulting in significantly decreased cell surface presentation of this HIV coreceptor.
This research elucidates the cytotoxic effects on human cells by immune activating IVT gRNAs in CRISPR-Cas9 or Cpf1 ribonucleoprotein complexes and offers a new method to prevent host immune response, while maintaining high mutation efficiency, toward the effective therapeutic application of CRISPR-mediated genome editing.
Researchers from the Institute for Basic Science, University of Science and Technology, National Medical Center, and Seoul National University contributed to this work. The study was funded by a grant from the Institute for Basic Science (IBS-R021-D1).
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About the article:
The manuscript will be published online ahead of print on 22 February 2018. Its full citation is as follows: Kim S, Koo T, Jee H, Cho H, Lee G, Lim D, Shik Shin H, and Kim J. 2018. CRISPR RNAs trigger innate immune responses in human cells. Genome Res doi: 10.1101/gr.231936.117
About Genome Research:
Launched in 1995, Genome Research is an international, continuously published, peer-reviewed journal that focuses on research that provides novel insights into the genome biology of all organisms, including advances in genomic medicine. Among the topics considered by the journal are genome structure and function, comparative genomics, molecular evolution, genome-scale quantitative and population genetics, proteomics, epigenomics, and systems biology. The journal also features exciting gene discoveries and reports of cutting-edge computational biology and high-throughput methodologies.
About Cold Spring Harbor Laboratory Press:
Cold Spring Harbor Laboratory Press is an internationally renowned publisher of books, journals, and electronic media, located on Long Island, New York. Since 1933, it has furthered the advance and spread of scientific knowledge in all areas of genetics and molecular biology, including cancer biology, plant science, bioinformatics, and neurobiology. The Press is a division of Cold Spring Harbor Laboratory, an innovator in life science research and the education of scientists, students, and the public. For more information, visit our website at http://cshlpress.
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