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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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Autores principales: Wienert, Beeke, Shin, Jiyung, Zelin, Elena, Pestal, Kathleen, Corn, Jacob E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049001/
https://www.ncbi.nlm.nih.gov/pubmed/30011268
http://dx.doi.org/10.1371/journal.pbio.2005840
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author Wienert, Beeke
Shin, Jiyung
Zelin, Elena
Pestal, Kathleen
Corn, Jacob E.
author_facet Wienert, Beeke
Shin, Jiyung
Zelin, Elena
Pestal, Kathleen
Corn, Jacob E.
author_sort Wienert, Beeke
collection PubMed
description 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.
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spelling pubmed-60490012018-07-26 In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway Wienert, Beeke Shin, Jiyung Zelin, Elena Pestal, Kathleen Corn, Jacob E. PLoS Biol Short Reports 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. Public Library of Science 2018-07-16 /pmc/articles/PMC6049001/ /pubmed/30011268 http://dx.doi.org/10.1371/journal.pbio.2005840 Text en © 2018 Wienert et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Short Reports
Wienert, Beeke
Shin, Jiyung
Zelin, Elena
Pestal, Kathleen
Corn, Jacob E.
In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway
title In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway
title_full In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway
title_fullStr In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway
title_full_unstemmed In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway
title_short In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway
title_sort in vitro–transcribed guide rnas trigger an innate immune response via the rig-i pathway
topic Short Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049001/
https://www.ncbi.nlm.nih.gov/pubmed/30011268
http://dx.doi.org/10.1371/journal.pbio.2005840
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