PubReading [27] - Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology - N. R. Hansmeier, J.W. Kornfeld et al.

<p>In recent years, long noncoding RNAs (<strong>lncRNA</strong>s) have emerged as multifaceted regulators of <strong>gene expression</strong>, 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<strong> (CRISPR)/Cas9 </strong>platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including <em>Caenorhabditis elegans</em>, <em>Drosophila melanogaster</em>, <em>Danio rerio </em>and <em>Mus musculus</em>. 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 <em>Gm15441</em>, 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