In vitro-transcribed guide RNAs trigger an innate immune response via the RIG-I pathway

PLoS Biol. 2018 Jul 16;16(7):e2005840. doi: 10.1371/journal.pbio.2005840. eCollection 2018 Jul.

Abstract

Clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While editing of immortalized cell lines has become relatively easy, editing of therapeutically relevant primary cells and tissues can remain challenging. One recent advancement is the delivery of a Cas9 protein and an in vitro-transcribed (IVT) guide RNA (gRNA) as a precomplexed ribonucleoprotein (RNP). This approach allows editing of primary cells such as T cells and hematopoietic stem cells, but the consequences beyond genome editing of introducing foreign Cas9 RNPs into mammalian cells are not fully understood. Here, we show that the IVT gRNAs commonly used by many laboratories for RNP editing trigger a potent innate immune response that is similar to canonical immune-stimulating ligands. IVT gRNAs are recognized in the cytosol through the retinoic acid-inducible gene I (RIG-I) pathway but not the melanoma differentiation-associated gene 5 (MDA5) pathway, thereby triggering a type I interferon response. Removal of the 5'-triphosphate from gRNAs ameliorates inflammatory signaling and prevents the loss of viability associated with genome editing in hematopoietic stem cells. The potential for Cas9 RNP editing to induce a potent antiviral response indicates that care must be taken when designing therapeutic strategies to edit primary cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cytosol / metabolism
  • DEAD Box Protein 58 / metabolism*
  • Humans
  • Immunity, Innate / genetics*
  • Interferon Type I / metabolism
  • Models, Biological
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism
  • Transcription, Genetic*

Substances

  • Interferon Type I
  • RNA, Guide
  • DDX58 protein, human
  • DEAD Box Protein 58

Grant support

Li Ka Shing Foundation. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Heritage Medical Research Institute. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH (grant number DP2-HL-141006). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.