CRISPR RNAs trigger innate immune responses in human cells

Author:

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

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.

Funder

Institute for Basic Science

Publisher

Cold Spring Harbor Laboratory

Subject

Genetics (clinical),Genetics

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