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Non-Coding RNA 2019, 5(1), 12; https://doi.org/10.3390/ncrna5010012

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

1
Max Planck Institute for Metabolism Research, Gleueler Strasse 50, 50931 Cologne, Germany
2
Cologne Cluster of Excellence: Cellular Stress Responses in Ageing-associated Diseases, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany
3
Institute for Prophylaxis and Epidemiology of Cardiovascular Diseases (IPEK), Ludwig Maximilian University of Munich, 80336 Munich, Germany
4
Department for Biochemistry and Molecular Biology, Functional Genomics and Metabolism Unit, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
*
Author to whom correspondence should be addressed.
Received: 9 November 2018 / Revised: 11 January 2019 / Accepted: 14 January 2019 / Published: 23 January 2019
(This article belongs to the Special Issue Non-Coding RNA and Diabetes)
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Abstract

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. View Full-Text
Keywords: long noncoding RNA; clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome engineering; knockout mice long noncoding RNA; clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome engineering; knockout mice
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Hansmeier, N.R.; Widdershooven, P.J.M.; Khani, S.; Kornfeld, J.-W. Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology. Non-Coding RNA 2019, 5, 12.

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