TY - UNPB
T1 - Transcript-Specific Site-Directed RNA Editing Reveals Principles for Splice-aware guide RNA Design
AU - Schneider, Nina
AU - Zehoray, Nave
AU - Steinberg, Ricky
AU - Banin, Eyal
AU - Arsenijevic, Yvan
AU - Ben Aroya, Shay
AU - Levanon, Erez Y.
AU - Sharon, Dror
PY - 2026/1/1
Y1 - 2026/1/1
N2 - Site-directed RNA editing (SDRE) utilizing the adenosine deaminase acting on RNA (ADAR) enzymes is commonly facilitated by guide RNAs (gRNAs) optimized to enhance on-target editing and minimize bystander effects. However, the impact of gRNA binding and ADAR-mediated SDRE on canonical pre-mRNA splicing remains poorly understood. Here, we developed an in vitro transcript-specific editing strategy that enables selective targeting and direct comparison of SDRE in pre-mRNA and mature mRNA. Using splice-relevant variants associated with inherited retinal diseases, we investigated the effects of SDRE on exonic, near-canonical intronic, and deep intronic splice variants. We identified gRNA-induced splice perturbation at exonic and intronic targets and observed that higher editing levels could be associated with increased splice disruption. Conversely, SDRE of two exonic splice variants and a deep intronic variant resulted in increased production of correctly spliced transcripts, demonstrating the potential of SDRE for splice modulation. Finally, by dissecting the effects of ADAR expression and gRNA design on editing and splicing outcomes, we established a system for identifying design principles that reduce splice interference and enhance the generation of correctly spliced, edited transcripts. These findings highlight the importance of considering transcript context and splicing consequences in the development of SDRE-based therapeutic strategies.Created in BioRender. Schneider, N. (2026) https://BioRender.com/59i0tiyCompeting Interest StatementThe authors have declared no competing interest.All processed data supporting the findings of this study are included in the article and its Supplementary Data. The source code used in this study (Figure 1 and Supplementary Table 1) is available on GitHub (https://github.com/znavezz/clinvar-ga-splicing) and is permanently archived in Zenodo at 10.5281/zenodo.21692997. Raw sequencing data, Sanger chromatograms, capillary electrophoresis data, and ddPCR output files are available from the corresponding author upon reasonable request.Israel Science Foundation, 566/23, 2637/23Foundation Fighting Blindness, PPA-0923-0865-HUJScience Training Encouraging Peace (STEP) fellowshipAriane de Rothschild Women Doctoral Program
AB - Site-directed RNA editing (SDRE) utilizing the adenosine deaminase acting on RNA (ADAR) enzymes is commonly facilitated by guide RNAs (gRNAs) optimized to enhance on-target editing and minimize bystander effects. However, the impact of gRNA binding and ADAR-mediated SDRE on canonical pre-mRNA splicing remains poorly understood. Here, we developed an in vitro transcript-specific editing strategy that enables selective targeting and direct comparison of SDRE in pre-mRNA and mature mRNA. Using splice-relevant variants associated with inherited retinal diseases, we investigated the effects of SDRE on exonic, near-canonical intronic, and deep intronic splice variants. We identified gRNA-induced splice perturbation at exonic and intronic targets and observed that higher editing levels could be associated with increased splice disruption. Conversely, SDRE of two exonic splice variants and a deep intronic variant resulted in increased production of correctly spliced transcripts, demonstrating the potential of SDRE for splice modulation. Finally, by dissecting the effects of ADAR expression and gRNA design on editing and splicing outcomes, we established a system for identifying design principles that reduce splice interference and enhance the generation of correctly spliced, edited transcripts. These findings highlight the importance of considering transcript context and splicing consequences in the development of SDRE-based therapeutic strategies.Created in BioRender. Schneider, N. (2026) https://BioRender.com/59i0tiyCompeting Interest StatementThe authors have declared no competing interest.All processed data supporting the findings of this study are included in the article and its Supplementary Data. The source code used in this study (Figure 1 and Supplementary Table 1) is available on GitHub (https://github.com/znavezz/clinvar-ga-splicing) and is permanently archived in Zenodo at 10.5281/zenodo.21692997. Raw sequencing data, Sanger chromatograms, capillary electrophoresis data, and ddPCR output files are available from the corresponding author upon reasonable request.Israel Science Foundation, 566/23, 2637/23Foundation Fighting Blindness, PPA-0923-0865-HUJScience Training Encouraging Peace (STEP) fellowshipAriane de Rothschild Women Doctoral Program
U2 - 10.64898/2026.07.30.741163
DO - 10.64898/2026.07.30.741163
M3 - Preprint
T3 - bioRxiv
SP - 2026.07.30.741163
BT - Transcript-Specific Site-Directed RNA Editing Reveals Principles for Splice-aware guide RNA Design
ER -