CRISPR RNAs trigger innate immune responses in human cells

Sojung Kim; Taeyoung Koo; Hyeon-Gun Jee; Hee-Yeon Cho; Gyeorae Lee; Dong-Gyun Lim; Hyoung Shik Shin; Jin-Soo Kim

doi:10.1101/gr.231936.117

CRISPR RNAs trigger innate immune responses in human cells

Genome Res. 2018 Feb 22;28(3):367-373. doi: 10.1101/gr.231936.117. Online ahead of print.

Authors

Sojung Kim^#¹, Taeyoung Koo^#^{1

2}, Hyeon-Gun Jee^#³, Hee-Yeon Cho¹, Gyeorae Lee^{1

2}, Dong-Gyun Lim³, Hyoung Shik Shin⁴, Jin-Soo Kim^{1

2

5}

Affiliations

¹ Center for Genome Engineering, Institute for Basic Science, Seoul 08826, South Korea.
² Department of Basic Science, University of Science & Technology, Daejeon 34113, South Korea.
³ Center for Chronic Diseases, Research Institute, National Medical Center, Seoul 04564, South Korea.
⁴ Research Center for Infectious Diseases, Research Institute, National Medical Center, Seoul 04564, South Korea.
⁵ Department of Chemistry, Seoul National University, Seoul 08826, South Korea.

^# Contributed equally.

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.