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Effective CRISPR interference of an endogenous gene via a single transgene in mice

Drawbacks of conditional gene deletion in mice include the need for extensive breeding and, often, a lack of cell type-specificity. CRISPR interference (CRISPRi) is an alternative approach for loss-of-function studies that inhibits expression by guiding a transcriptional repressor to the transcripti...

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Autores principales: MacLeod, Ryan S., Cawley, Keisha M., Gubrij, Igor, Nookaew, Intawat, Onal, Melda, O’Brien, Charles A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872636/
https://www.ncbi.nlm.nih.gov/pubmed/31754144
http://dx.doi.org/10.1038/s41598-019-53611-6
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author MacLeod, Ryan S.
Cawley, Keisha M.
Gubrij, Igor
Nookaew, Intawat
Onal, Melda
O’Brien, Charles A.
author_facet MacLeod, Ryan S.
Cawley, Keisha M.
Gubrij, Igor
Nookaew, Intawat
Onal, Melda
O’Brien, Charles A.
author_sort MacLeod, Ryan S.
collection PubMed
description Drawbacks of conditional gene deletion in mice include the need for extensive breeding and, often, a lack of cell type-specificity. CRISPR interference (CRISPRi) is an alternative approach for loss-of-function studies that inhibits expression by guiding a transcriptional repressor to the transcription start-site of target genes. However, there has been limited exploration of CRISPRi in mice. We tested the effectiveness of a single CRISPRi transgene broadly expressing a single guide RNA and a catalytically dead Cas9 fused to the KRAB repressor domain to suppress a well-characterized target gene, Tnfsf11. The phenotype of CRISPRi transgenic mice was compared to mice with germline deletion of Tnfsf11, which are osteopetrotic and do not form lymph nodes. High transgene expression mimicked gene deletion, with failure of lymph node development and classic signs of osteopetrosis such as high bone mass and failure of tooth eruption. Mice with low transgene expression were normal and mice with medium expression displayed an intermediate phenotype. Transgene expression in tissues from these mice correlated inversely with Tnfsf11 mRNA levels. These results demonstrate that a single CRISPRi transgene can effectively suppress a target gene in mice and suggest that this approach may be useful for cell type-specific loss-of-function studies.
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spelling pubmed-68726362019-12-04 Effective CRISPR interference of an endogenous gene via a single transgene in mice MacLeod, Ryan S. Cawley, Keisha M. Gubrij, Igor Nookaew, Intawat Onal, Melda O’Brien, Charles A. Sci Rep Article Drawbacks of conditional gene deletion in mice include the need for extensive breeding and, often, a lack of cell type-specificity. CRISPR interference (CRISPRi) is an alternative approach for loss-of-function studies that inhibits expression by guiding a transcriptional repressor to the transcription start-site of target genes. However, there has been limited exploration of CRISPRi in mice. We tested the effectiveness of a single CRISPRi transgene broadly expressing a single guide RNA and a catalytically dead Cas9 fused to the KRAB repressor domain to suppress a well-characterized target gene, Tnfsf11. The phenotype of CRISPRi transgenic mice was compared to mice with germline deletion of Tnfsf11, which are osteopetrotic and do not form lymph nodes. High transgene expression mimicked gene deletion, with failure of lymph node development and classic signs of osteopetrosis such as high bone mass and failure of tooth eruption. Mice with low transgene expression were normal and mice with medium expression displayed an intermediate phenotype. Transgene expression in tissues from these mice correlated inversely with Tnfsf11 mRNA levels. These results demonstrate that a single CRISPRi transgene can effectively suppress a target gene in mice and suggest that this approach may be useful for cell type-specific loss-of-function studies. Nature Publishing Group UK 2019-11-21 /pmc/articles/PMC6872636/ /pubmed/31754144 http://dx.doi.org/10.1038/s41598-019-53611-6 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
MacLeod, Ryan S.
Cawley, Keisha M.
Gubrij, Igor
Nookaew, Intawat
Onal, Melda
O’Brien, Charles A.
Effective CRISPR interference of an endogenous gene via a single transgene in mice
title Effective CRISPR interference of an endogenous gene via a single transgene in mice
title_full Effective CRISPR interference of an endogenous gene via a single transgene in mice
title_fullStr Effective CRISPR interference of an endogenous gene via a single transgene in mice
title_full_unstemmed Effective CRISPR interference of an endogenous gene via a single transgene in mice
title_short Effective CRISPR interference of an endogenous gene via a single transgene in mice
title_sort effective crispr interference of an endogenous gene via a single transgene in mice
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872636/
https://www.ncbi.nlm.nih.gov/pubmed/31754144
http://dx.doi.org/10.1038/s41598-019-53611-6
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