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CRISPR RNAs trigger innate immune responses in human cells

Cited 49 time in Web of Science Cited 53 time in Scopus
Authors
Kim, Sojung; Koo, Taeyoung; Jee, Hyeon-Gun; Cho, Hee-Yeon; Lee, Gyeorae; Lim, Dong-Gyun; Shin, Hyoung Shik; Kim, Jin-Soo
Issue Date
2018-03
Citation
Genome Research, Vol.28 No.3, pp.367-373
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 similar to 80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting St-hydroxyl gRNAs in complex with Cas9 or Cpfl 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.
ISSN
1088-9051
URI
http://hdl.handle.net/10371/165708
DOI
https://doi.org/10.1101/gr.231936.117
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