%0 Journal Article
%T Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology
%A Jan-Wilhelm Kornfeld
%A Nils R. Hansmeier
%A Pia J. M. Widdershooven
%A Sajjad Khani
%J Archive of "Non-Coding RNA".
%D 2019
%R 10.3390/ncrna5010012
%X 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¨C20 weeks, of which <10 days hands-on working time is required
%K long noncoding RNA
%K clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome engineering
%K knockout mice
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468733/