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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| Format: | Article |
| Language: | English |
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MDPI AG
2019-01-01
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| Series: | Non-Coding RNA |
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| Online Access: | https://www.mdpi.com/2311-553X/5/1/12 |
| _version_ | 1818539336229978112 |
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| author | Nils R. Hansmeier Pia J. M. Widdershooven Sajjad Khani Jan-Wilhelm Kornfeld |
| author_facet | Nils R. Hansmeier Pia J. M. Widdershooven Sajjad Khani Jan-Wilhelm Kornfeld |
| author_sort | Nils R. Hansmeier |
| collection | DOAJ |
| 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 <i>Caenorhabditis elegans</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i> and <i>Mus musculus</i>. 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 <i>Gm15441</i>, 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. |
| first_indexed | 2024-12-11T21:40:41Z |
| format | Article |
| id | doaj.art-f804dd4eaf644ae7a8d9abf9fe522315 |
| institution | Directory Open Access Journal |
| issn | 2311-553X |
| language | English |
| last_indexed | 2024-12-11T21:40:41Z |
| publishDate | 2019-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Non-Coding RNA |
| spelling | doaj.art-f804dd4eaf644ae7a8d9abf9fe5223152022-12-22T00:49:51ZengMDPI AGNon-Coding RNA2311-553X2019-01-01511210.3390/ncrna5010012ncrna5010012Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 TechnologyNils R. Hansmeier0Pia J. M. Widdershooven1Sajjad Khani2Jan-Wilhelm Kornfeld3Max Planck Institute for Metabolism Research, Gleueler Strasse 50, 50931 Cologne, GermanyMax Planck Institute for Metabolism Research, Gleueler Strasse 50, 50931 Cologne, GermanyMax Planck Institute for Metabolism Research, Gleueler Strasse 50, 50931 Cologne, GermanyMax Planck Institute for Metabolism Research, Gleueler Strasse 50, 50931 Cologne, GermanyIn 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 <i>Caenorhabditis elegans</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i> and <i>Mus musculus</i>. 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 <i>Gm15441</i>, 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.https://www.mdpi.com/2311-553X/5/1/12long noncoding RNA, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome engineering, knockout mice |
| spellingShingle | Nils R. Hansmeier Pia J. M. Widdershooven Sajjad Khani Jan-Wilhelm Kornfeld Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology Non-Coding RNA long noncoding RNA, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome engineering, knockout mice |
| 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 | long noncoding RNA, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome engineering, knockout mice |
| url | https://www.mdpi.com/2311-553X/5/1/12 |
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