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Off-target effects of CRISPRa on interleukin-6 expression
Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816553/ https://www.ncbi.nlm.nih.gov/pubmed/31658298 http://dx.doi.org/10.1371/journal.pone.0224113 |
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author | Soubeyrand, Sébastien Lau, Paulina Peters, Victoria McPherson, Ruth |
author_facet | Soubeyrand, Sébastien Lau, Paulina Peters, Victoria McPherson, Ruth |
author_sort | Soubeyrand, Sébastien |
collection | PubMed |
description | Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology. |
format | Online Article Text |
id | pubmed-6816553 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-68165532019-11-03 Off-target effects of CRISPRa on interleukin-6 expression Soubeyrand, Sébastien Lau, Paulina Peters, Victoria McPherson, Ruth PLoS One Research Article Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology. Public Library of Science 2019-10-28 /pmc/articles/PMC6816553/ /pubmed/31658298 http://dx.doi.org/10.1371/journal.pone.0224113 Text en © 2019 Soubeyrand 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 | Research Article Soubeyrand, Sébastien Lau, Paulina Peters, Victoria McPherson, Ruth Off-target effects of CRISPRa on interleukin-6 expression |
title | Off-target effects of CRISPRa on interleukin-6 expression |
title_full | Off-target effects of CRISPRa on interleukin-6 expression |
title_fullStr | Off-target effects of CRISPRa on interleukin-6 expression |
title_full_unstemmed | Off-target effects of CRISPRa on interleukin-6 expression |
title_short | Off-target effects of CRISPRa on interleukin-6 expression |
title_sort | off-target effects of crispra on interleukin-6 expression |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816553/ https://www.ncbi.nlm.nih.gov/pubmed/31658298 http://dx.doi.org/10.1371/journal.pone.0224113 |
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