Abstract
Long noncoding RNAs (lncRNAs) are fundamental genomic regulatory factors under various physiological and pathological conditions. A class of lncRNAs termed architectural RNAs (arcRNAs) plays an essential scaffolding role in building nuclear bodies. NEAT1 arcRNA is an abundant, nuclear-retained lncRNA that constructs paraspeckle nuclear bodies. NEAT1 is upregulated in various developmental and disease conditions including cancer and virus infection. However, it remains unclear how elevated expression of NEAT1 influences such conditions. Here, we set up an experimental method to selectively increase NEAT1 expression. We applied the synergistic activation mediator (SAM) system using catalytically dead Cas9 (dCas9) proteins to activate transcription of the NEAT1 gene. We examined 10 pre-designed and 15 originally designed single-guide RNAs (sgRNAs) in the NEAT1 promoter region for CRISPR activation (CRISPRa). We validated several sgRNAs that we designed for the SAM system to strongly activate NEAT1 expression in two human cell lines and induced formation of paraspeckles with intact core-shell structures. Thus, this selective NEAT1 upregulation method using the SAM system would be useful for further functional analyses of NEAT1 lncRNA in both basic and applied research.
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