Abstract 2393: The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique

Abstract

Abstract Long non-coding RNAs (LncRNAs) have been found to play important roles in maintaining and promoting cellular proliferation in cancerous cells. One such lncRNA, LINC00963 has been found to promote cellular proliferation, migration and tumorigenesis in prostate, ovarian, breast and non-small cell lung (NSCLC) cancer. In this study, we aim to identify important functional regions of this lncRNA with a view to identify unique regions for targeted drug therapy. In order to achieve this, we sought to investigate this lncRNA in the A549 NSCLC cell line. We found that LINC00963 was highly expressed and when targeted with either siRNAs or antisense oligonucleotides, cells rapidly undergo apoptosis. Using cell death as a proxy for loss-of-function, we characterized LINC00963 at the genomic level using a CRIPSRScan approach. CRISPRScan is a novel way of determining lncRNA functional domains through the introduction of local mutations. These mutations could potentially disrupt lncRNA function, which for LINC00963 would lead to apoptosis of the cell. We created a unique lentivirus library comprised of 1784 single guide RNAs spanning the length of LINC00963 in order to precisely identify regions of functional importance. Upon transduction in a stably expressing Cas9-A549 cell line, we collected cellular fractions over time, and using high-throughput sequencing (NextSeq, Illumina), evaluated the depletion of these sgRNAs as a proxy for their importance in LINC00963 function. The identification of these key genomic regions that may render LINC00963 non-functional may offer new lncRNA target sites for anti-cancer therapies in the future. Additionally, the utilization of an in-depth CRISPR-Cas9 approach to determine important lncRNA domains provides a novel and precise way in which to evaluate lncRNA function. Citation Format: Roslyn M. Ray, Lu Lang, Sheela P. Pokharel, Denis O'Meally, Tristan A. Scott, Chun-Wei Chen, Kevin V. Morris. The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2393.