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

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

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Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, United States of America.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States of America.


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.

Conflict of interest statement

I have read the journal's policy, and the authors of this manuscript have the following competing interests. JEC is a founder of Spotlight Therapeutics and Peregrine Biotechnology. JEC is also on the Scientific Advisory Board of Inari Agriculture and Mission Biotechnology. JEC's lab has unrelated scientific collaborations with AstraZeneca and Agilent Technologies.

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