225-LB: A High-Throughput CRISPR-Activation Screen Defines Human LncRNA Metabolic Regulators

In the past decade, long noncoding RNAs (lncRNAs) have emerged as important regulators of cellular and systemic metabolism. Deciphering the physiological functions of lncRNA by gain- or loss-of-function analyses in key metabolic organs, such as the liver, is essential to understand their disease relevance. Conventional transgenic or knockout approaches, however, have limitations in defining lncRNA function as lncRNAs transgenically expressed from ectopic genomic loci could be non-functional and partial deletion of lncRNAs. Domain negative Cas9 (dCas9) coupled with transcriptional activators can trigger endogenous lncRNA expression by targeting guide RNA (gRNA) to transcription start sites of lncRNAs, thus increasing their transcription efficiency and targeting multiple different lncRNA isoforms simultaneously. Adenovirus vector is the most commonly used tool for specifically targeting liver-enriched genes both in vitro and in animal models. A CRISPR-activation (CRISPR-a) adenovirus system thus constitutes a powerful means to active liver lncRNAs transcriptionally. In this study, we established a CRISPR-a adenovirus platform by transferring CRISPR-a units into the adenovirus vector, and our system allows one digestion-ligation step to construct the gRNA adenovirus vector seamlessly. Efficacy and versatility of this pipeline has been confirmed by the successful construction of a CRISPR-a adenovirus library containing 96 gRNAs targeting 20 liver-enriched, metabolic sensitive lncRNAs, which shows high CRISPR activation efficiency after primary human hepatocytes infection. Moreover, the expression levels of several lncRNA target genes predicted by co-relation analysis are significantly changed. This method thus provides a fast and efficient way to activate the endogenous liver lncRNAs and simplifies the procedure of lncRNA functional screening using CRISPR-a adenovirus system, which can be scaled up to a genome level to quickly define lncRNA metabolic regulators.