In:
Non-Coding RNA, MDPI AG, Vol. 5, No. 1 ( 2019-01-23), p. 12-
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.
Type of Medium:
Online Resource
ISSN:
2311-553X
DOI:
10.3390/ncrna5010012
Language:
English
Publisher:
MDPI AG
Publication Date:
2019
detail.hit.zdb_id:
2813993-8
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