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

Nils R Hansmeier, Pia J M Widdershooven, Sajjad Khani, Jan-Wilhelm Kornfeld

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

171 Downloads (Pure)

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.

OriginalsprogEngelsk
Artikelnummer12
TidsskriftNon-Coding R N A
Vol/bind5
Udgave nummer1
Sider (fra-til)1-12
ISSN2311-553X
DOI
StatusUdgivet - 23. jan. 2019

Fingeraftryk

Dyk ned i forskningsemnerne om 'Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology'. Sammen danner de et unikt fingeraftryk.

Citationsformater