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

Research output: Contribution to journalArticlepeer-review

171 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)367-373
Number of pages7
JournalGenome Research
Volume28
Issue number3
DOIs
Publication statusPublished - Mar 2018

Bibliographical note

Publisher Copyright:
© 2018 Kim et al.

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