Abstract
Purpose :
Single nucleotide editing can be performed at the RNA level using the human deaminase acting on RNA (ADAR) enzyme and can serve as a tool for gene therapy of inherited diseases, including inherited retinal diseases (IRDs). ADAR-based RNA editing requires the delivery of an efficient guideRNA (gRNA) that is designed to recruit the endogenously expressed ADAR enzyme to a mutated RNA. Our aim is to design and test gRNAs that induce targeted ADAR editing for relatively common IRD-causing mutations including nonsense, missense, and splice-site mutations.
Methods :
A yeast model was used to identify candidate gRNAs for nonsense mutations by measuring yeast survival and percent editing using next generation sequencing (NGS). A fluorescence-expressing plasmid reporter system was used in HeLa cells overexpressing either ADAR1/2. The cells were transfected by a plasmid that includes a gene cassette harboring the studied mutation in between mCherry and EGFP. Splicing efficiency was measured by gel electrophoresis of PCR products as well as next generation sequencing (NGS) analysis.
Results :
RNA editing of the following mutations was studied: USH2A-c.11864G>A (p.W3955*), TRPM1-c.880A>T (p.K294*), GUCY2D-c.2513G>A (p.R838H), and NR2E3-c.932G>A (p.R311Q) in HeLa cells. The analysis yielded average editing levels of 27%, 39%, 12%, and 0%, respectively. Aiming to better characterize the interplay between RNA editing and splicing, we further studied RNA editing of the TRPM1 mutation that is located in proximity at an exon-intron boundary. Two gRNAs were designed for TRPM1, an exon-exon and an exon-intron gRNA targeting the mRNA and pre-mRNA molecules and yielded editing levels of 37% and 32% respectively. Our results show that the exon-intron gRNA does not prevent proper splicing. NGS analysis showed that pre-mRNA transcripts were rare but have higher editing levels when treated by exon-intron gRNA.
Conclusions :
A large variability in editing levels is observed between the various mutations. Studies of gRNA libraries in either yeasts or human cells are therefore needed to improve editing levels. No studies have been reported so far in which the effect of splicing on RNA editing of disease-causing mutations has been examined and therefore this study will shed light on editing efficiency and effect on splicing when performing ADAR-mediated RNA editing on areas close to or within splice sites.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.