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

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 therapeuti...

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Bibliographic Details
Published in:PLoS biology 2018-07, Vol.16 (7), p.e2005840
Main Authors: Wienert, Beeke, Shin, Jiyung, Zelin, Elena, Pestal, Kathleen, Corn, Jacob E
Format: Article
Language:English
Subjects:
Apoptosis
Biology
Biology and Life Sciences
Cell Line
Cell lines
CRISPR
Cytosol
Cytosol - metabolism
DEAD Box Protein 58 - metabolism
Deoxyribonucleic acid
DNA
Editing
Genes
Genetic engineering
Genome editing
Genomes
Genomics
gRNA
Hematopoietic stem cells
Humans
Immune response
Immune system
Immunity, Innate - genetics
Inflammation
Innate immunity
Interferon
Interferon Type I - metabolism
Ligands
Lymphocytes
Lymphocytes T
Mammalian cells
Medical treatment
Medicine and Health Sciences
Melanoma
Models, Biological
Plasmids
Proteins
Receptors, Immunologic
Research and Analysis Methods
Retinoic acid
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
Short Reports
Stem cells
Transcription, Genetic
Viability
Viruses
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Summary: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.
ISSN:1545-7885
1544-9173
1545-7885
DOI:10.1371/journal.pbio.2005840