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Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in orga...

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Autores principales: Hansmeier, Nils R., Widdershooven, Pia J. M., Khani, Sajjad, Kornfeld, Jan-Wilhelm
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468733/
https://www.ncbi.nlm.nih.gov/pubmed/30678101
http://dx.doi.org/10.3390/ncrna5010012
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author Hansmeier, Nils R.
Widdershooven, Pia J. M.
Khani, Sajjad
Kornfeld, Jan-Wilhelm
author_facet Hansmeier, Nils R.
Widdershooven, Pia J. M.
Khani, Sajjad
Kornfeld, Jan-Wilhelm
author_sort Hansmeier, Nils R.
collection PubMed
description In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required.
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spelling pubmed-64687332019-04-19 Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology Hansmeier, Nils R. Widdershooven, Pia J. M. Khani, Sajjad Kornfeld, Jan-Wilhelm Noncoding RNA Article In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required. MDPI 2019-01-23 /pmc/articles/PMC6468733/ /pubmed/30678101 http://dx.doi.org/10.3390/ncrna5010012 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hansmeier, Nils R.
Widdershooven, Pia J. M.
Khani, Sajjad
Kornfeld, Jan-Wilhelm
Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology
title Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology
title_full Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology
title_fullStr Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology
title_full_unstemmed Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology
title_short Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology
title_sort rapid generation of long noncoding rna knockout mice using crispr/cas9 technology
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468733/
https://www.ncbi.nlm.nih.gov/pubmed/30678101
http://dx.doi.org/10.3390/ncrna5010012
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