Poster abstracts
Poster number 60 submitted by Shashi Singh
Engineered circ-arRNAs enable RNA editing and splice modulation for tunable oncogene silencing
Shashi S. Singh (Department of Biochemistry and Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Joseph M. Luna (Department of Biochemistry and Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)
Abstract:
RNA editing provides a reversible and precise approach for gene modulation, offering advantages over permanent genome editing. We present a novel platform using circular ADAR-recruiting RNAs (circ-arRNAs) to harness endogenous Adenosine Deaminase Acting on RNA (ADAR) activity for targeted adenosine-to-inosine (A-to-I) editing. These self-cleaving, self-ligating RNAs form stable circular structures capable of guiding ADAR to specific transcripts. We demonstrate the utility of circ-arRNAs in removing a stop codon (UAG→UIG) in a dual fluorescent reporter as well as correcting the cancer-driving mutation KRAS G13D by editing (GAC→GIC) to the wild-type sequence. After establishing successful editing in both applications, we turned to conserved adenosines at 3′splice sites. In particular, we focused on exon 3 of KRAS, where skipping introduces an out-of-frame splice and destabilizes the mRNA and found that circ-arRNAs consistently induced exon 3 skipping accompanied by reduced KRAS protein expression. Unexpectedly, these effects persisted even in ADAR-deficient cells, suggesting an additional antisense oligonucleotide (ASO)-like activity that is independent of RNA editing. Using a compact 111-nucleotide design, we tiled guides across exon-intron boundaries of KRAS 3 and observed that only guides engaging exonic regions, including junctions, elicited efficient skipping, whereas intron-restricted guides were ineffective. Together, these findings establish circ-arRNAs as a dual-function platform that couples programmable RNA editing with ASO-like splice modulation, offering strong therapeutic potential for transient and reversible gene silencing in cancer and other diseases.
Keywords: RNA Editing, Circular RNA, RNA Splicing and Cancer