Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Abstract

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Figure 1:. A dual vector system to overexpress the lncRNA IFNG-AS1.

(A) A schematic of the IFNG-AS1 gene structure and the relationship between guide RNA (gRNA) binding sites and the transcriptional start site (TSS). (B) The features of the gRNA and dCAS9 vectors.

Figure 2:. Viral titering and colony screening.

(A) An example p24 ELISA standard curve for lentivirus-containing, conditioned media. The black dots represent standard samples and the red dots unknown samples. (B) After transducing, selecting, and generating colonies, real-time PCR and gel electrophoresis against dCas9 were performed on the RNA from either nontransduced Jurkat cells (parental) or dCas9-transduced clones. No reverse transcriptase (No RT) controls were performed.

Figure 3:. Measuring IFNG-AS1 gene expression.

(A) A diagram of the relationship between primer sets and transcript variants. The red arrows represent transcript-specific primers, while the blue arrows indicate primers against all known transcripts. (B and C) Representative average PCR curves and fold-change quantifications for control and IFNG-AS1-overexpressing cells. RFU = relative fluorescence units. N = 3 samples per group. Mean ± SD. *p < 0.05, ***p < 0.001. A Student’s t-test was used to calculate the p-values.