Abstract 13395: Identification of Regulatory Regions of lncRNA Malat 1 Using CRISPR/Cas9-Based Screening Approaches

The cardiovascular system plays an essential role in regulating and maintaining organ function in the human body. The advances in sequencing technology led to the identification of thousands of non-coding RNAs, which functions by regulating biological processes by various mechanisms. MALAT1 has been among the first described long non-coding RNAs that was shown to regulate angiogenesis, however, the mechanisms underlying MALAT1 effects in endothelial cells remain elusive. To identify regulatory region in the ~8.7 kb MALAT1 transcript, we used an innovative, unbiased screening approach based on 3Cs-gRNAs (Covalently-Closed-Circular synthesized (3Cs)) technology that permitted us the generation of CRISPR/Cas gRNA library that contain all of the possible gRNA’s for non-coding RNA MALAT1. Using overlapping-oligo’s synthesis, we generated a pool of 998 gRNA which cover the whole MALAT1 sequence. Human endothelial cells (EC) were transduced with lentivirus particles harboring the gRNA pool. As silencing of MALAT1 leads to inhibition of EC proliferation, ECs were cultured and passaged for 14 days followed by sequencing of the gRNAs. Sequences were compared to the originally transduced ECs to identify those sequences, which were depleted during the passages.12 sequences, which were significantly depleted were functionally validated. 6 of gRNA candidates, among them 4 within a regulatory regions (spanning a stretch of MALAT1 RNA between nucleotides 3000 and 4000) induced a cell cycle arrest (Fig.1) and inhibited ECs viability (from 25 to 42% reduction, p