Abstract 216: CRISPR screen validates RIC-seq revealed eRNA-uaRNA interactions and transcriptome changes in gastric cancer

Abstract Introduction: Enhancer RNA (eRNA) is generated by the transcription of activated enhancers and belongs to non-coding RNA, regulating epigenetic activities and significantly affecting the transcription and activating target genes. We use RIC-seq (RNA in situ conformation sequencing) to simultaneously identify RNA-RNA interactions and loop structure between enhancers and promoters in the genome. RIC-seq also characterizes hubRNAs, which are transcribed from motifs similar to hubs that interact with globally distinct RNA fragments and transcriptionally robust and structurally conserved. Methods: We performed RIC-seq analysis of GC cell lines and normal gastric mucosa cell lines to capture RNA high-level structures, and identify sites of molecular enhancer RNA-uaRNA (promoter region transcribed RNAs) interactions and annotation information at the cellular in situ level. Several CRISPR libraries, including CRISPRa/i and CRISPR-Cas13 libraries were designed to validate these interactions in two GC cell lines. CROP-seq was used to identify transcriptome-level expression patterns generated from eRNA or uaRNA changes. Results: Based on RIC-seq, we identified direct or RBP-mediated indirect RNA-RNA proximity interactions and global enhancer-promoter loops among GC chromatin structure and specific spatial contacts. We found 88,696 significant pairs of RNA-RNA interactions in the GC cell line, while 96,990 significant pairs in the normal gastric mucosa cell line. Then, 831 pairs of RNA-RNA interactions and 146 eRNA-uaRNA interactions showed significantly differential expression between such two cell lines. All hubRNAs matched by RIC-seq were categorized and demonstrated that hubRNAs were mainly concentrated in protein-coding RNAs, antisense RNAs, and lincRNAs. From all these differentially expressed interactions, we constructed CRISPRa/i libraries using 10 relatively high expressed eRNAs and 116 target genes corresponding to uaRNAs respectively. Three hubRNAs and thirty-eight eRNAs were selected to construct CRISPR-Cas13 library for further validation of interested interactions. We found and validated eRNAs and target genes which were significantly related to GC cell proliferation, and resistance of target genes of 5-Fluorouracil (5-FU) and Oxaliplatin (Oxa). Through CROP-seq, we obtained transcriptome changes at the single-cell level caused by changes in target genes, further revealing the mechanisms underlying the influence of eRNA-uaRNA interactions on transcriptional regulation. Conclusion: We explored the mechanism of enhancer-promoter interactions by capturing all direct eRNA-uaRNA pairs in GC obtained by RIC-seq, and validation by CRISPR library screen combined with CROP-seq, yielding potential biomarkers and therapeutic targets in GC treatment. Citation Format: Xiangnan Zhang, Yanxin Yao, Hongji Dai, Wei Wang, Kexin Chen, Ben Liu. CRISPR screen validates RIC-seq revealed eRNA-uaRNA interactions and transcriptome changes in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 216.