High levels of ADAR overexpression induce abundant and stochastic off-target RNA editing in rice protoplasts

This study was led by Professor Kabin Xie (National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China). The authors found that overexpression of ADARdd in rice protoplasts leads to abundant off-target editing, a phenomenon not observed in leaves of stable transgenic rice plants overexpressing ADARdd (ADAR deaminase domain). To investigate the underlying mechanisms, the authors systematically explored the effects of ADARdd expression levels, protoplast physiology, and the presence of RNA-binding domains or a nuclear localization signal (NLS) on off-target editing. The results indicated that off-target editing by ADARdd was independent of protoplast physiology. Furthermore, the introduction of an RNA-binding domain or NLS also failed to prevent off-target activity in protoplasts transient expression. Further analysis indicated that the high levels of ADARdd overexpression alone is sufficient to cause global off-target editing.

  The authors further demonstrated that ADARdd-mediated off-target editing is characterized by pronounced stochasticity and low efficiency. The off-target editing sites (OFTEs) detected in protoplasts exhibited an even distribution across mRNAs, with editing efficiencies generally below 40%. In contrast, high-confidence editing sites identified in leaves of OsDRB1-ADARdd overexpressing plants (Yin et al., 2023) were significantly enriched in untranslated regions (UTRs), with efficiencies ranging from 40% to 100%. Further analysis of transcriptome data established an empirical expression threshold for ADARdd (log₂[TPM] < 13) to minimize off-target editing and identified several endogenous promoters that might exceed this threshold. This analysis suggested that ADARdd expression driven by the rice UBIQUITIN promoter remained below the threshold in stable transgenic plants, resulting in minimal off-target effects. However, several stress-inducible promoters, including the OsSalT and OCPI2 promoters, can exceed this threshold under stress conditions and thus require careful validation for use in RNA editing applications.

This study provides a comprehensive evaluation of the off-target effects of ADARdd and provides guidelines for improving RNA editing accuracy in plants. The authors also emphasize that protoplast-based assessments of proximity-directed RNA editors require rigorous validation.

See the article:

High levels of ADAR overexpression induce abundant and stochastic off-target RNA editing in rice protoplasts

https://www.sciencedirect.com/science/article/pii/S2662173825002140