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.
Highlights
A rather unexpected finding from Next-Generation RNA Sequencing (RNA-Seq) initiatives such as ENCODE [1], FANTOM [2] and NONCODE [3] was the observation that, whilst only two percent of genomes in higher organisms encode for protein-coding genes, more than two-thirds are transcribed across developmental stages and cell types [4]
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9) system has endowed us with a versatile platform, where the endonuclease Cas9 is recruited to specific genomic sites by virtue of a sequence-dependent CRISPR RNA and a sequence-independent trans-activating CRISPR RNA [15,16]—two RNA molecules that can be fused to a so-called single guide RNA for simplicity reasons [17]
One can validate long noncoding RNAs (lncRNAs) targeting efficiency in vitro within 2 weeks, and can successfully generate lncRNA-deficient mouse lines within 4 months. This protocol is broken up into four parts, including lncRNA selection (Step 1), validation of single guide RNA (sgRNA) efficacy in vitro (Step 2), sgRNA–Cas9 ribonucleoprotein (RNP) assembly and pronuclear microinjection (Step 3), and validation of lncRNA-null founder animals followed by breeding with C57BL/6 mice for germline transmission (Step 4)
Summary
A rather unexpected finding from Next-Generation RNA Sequencing (RNA-Seq) initiatives such as ENCODE (encyclopedia of DNA elements) [1], FANTOM (functional annotation of the mammalian genome) [2] and NONCODE (an integrated knowledge database dedicated to ncRNAs) [3] was the observation that, whilst only two percent of genomes in higher organisms encode for protein-coding genes, more than two-thirds are transcribed across developmental stages and cell types [4] This discovery led to the identification of several thousand so-called long noncoding RNAs (lncRNAs) [5,6] in mice and humans [7]. These procedures can take up to several months to generate a correctly targeted ES cell clone and a homozygous transgenic mouse line, whereas our methodology can generate loss-of-function mouse models within 18–20 weeks
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