CRISPR RNAs trigger innate immune responses in human cells
- Sojung Kim1,6,
- Taeyoung Koo1,2,6,
- Hyeon-Gun Jee3,6,
- Hee-Yeon Cho1,
- Gyeorae Lee1,2,
- Dong-Gyun Lim3,
- Hyoung Shik Shin4 and
- Jin-Soo Kim1,2,5
- 1Center for Genome Engineering, Institute for Basic Science, Seoul 08826, South Korea;
- 2Department of Basic Science, University of Science & Technology, Daejeon 34113, South Korea;
- 3Center for Chronic Diseases, Research Institute, National Medical Center, Seoul 04564, South Korea;
- 4Research Center for Infectious Diseases, Research Institute, National Medical Center, Seoul 04564, South Korea;
- 5Department of Chemistry, Seoul National University, Seoul 08826, South Korea
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↵6 These authors contributed equally to this work.
Abstract
Here, we report that CRISPR guide RNAs (gRNAs) with a 5′-triphosphate group (5′-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5′-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5′-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5′-hydroxyl group are much more efficient than in vitro–transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.231936.117.
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Freely available online through the Genome Research Open Access option.
- Received November 2, 2017.
- Accepted January 15, 2018.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
