Current methods for programmed RNA editing using endogenous ADAR enzymes and engineered ADAR-recruiting RNAs (arRNAs) suffer from low efficiency and bystander off-target editing. Here, we describe LEAPER 2.0, an updated version of LEAPER that uses covalently closed circular arRNAs, termed circ-arRNAs. We demonstrate on average ~3.1-fold higher editing efficiency than their linear counterparts when expressed in cells or delivered as in vitro-transcribed circular RNA oligonucleotides. To lower off-target editing we deleted pairings of uridines with off-target adenosines, which almost completely eliminated bystander off-target adenosine editing. Engineered circ-arRNAs enhanced the efficiency and fidelity of editing endogenous CTNNB1 and mutant TP53 transcripts in cell culture. Delivery of circ-arRNAs using adeno-associated virus in a mouse model of Hurler syndrome corrected the pathogenic point mutation and restored α-L-iduronidase catalytic activity, lowering glycosaminoglycan accumulation in the liver. LEAPER 2.0 provides a new design of arRNA that enables more precise, efficient RNA editing with broad applicability for therapy and basic research.
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Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
Nature Communications Open Access 13 January 2023
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All data and materials presented in this manuscript are available from the corresponding author (W.W.) upon reasonable request with a completed material transfer agreement. Raw data for whole-transcriptome RNA-seq are available as a BioProject with Project ID PRJNA775856. Source data are provided with this paper.
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We thank the staff of the BIOPIC High-throughput Sequencing Center (Peking University) and Genetron Health for their assistance in NGS analysis, and the National Center for Protein Sciences (Beijing) and the flow cytometry Core at National Center for Protein Sciences at Peking University, particularly Y. Guo and F. Wang, for technical help. We thank the High-Performance Computing Platform at Peking University for providing the platforms for NGS data analysis. We thank the Laboratory Animal Center at Peking University for the feeding of mice. This project was supported by funds from the National Key R&D Program of China (no. 2020YFA0707800), the Beijing Municipal Science & Technology Commission (no. Z181100001318009), the National Science Foundation of China (no. 31930016), the Beijing Advanced Innovation Center for Genomics at Peking University and the Peking-Tsinghua Center for Life Sciences (both to W.W.) and the Fellowship of China National Postdoctoral Program for Innovative Talents (no. BX20200010 to L.Q.).
Two patents have been filed relating to the data presented. W.W., Z.Y., L.Q., F.T. and C.W. are coinventors on patent applications describing circ-arRNA. W.W. and Z.Y. are coinventors on patent applications describing engineered circ-arRNA. P.Y., Z.Y. and Y.Z. are employees of EdiGene Inc. W.W. is a founder and scientific adviser for EdiGene, Inc. The other authors declare no competing interests.
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Supplementary Figs. 1–10.
Supplementary Table 1
Sequencing of arRNAs.
Supplementary Table 2
Sequencing of primers.
Source Data Fig. 1
Unprocessed immunoblots for Fig. 4e.
Source Data Fig. 2
Unprocessed gels for Supplementary Fig. 2e.
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Yi, Z., Qu, L., Tang, H. et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nat Biotechnol 40, 946–955 (2022). https://doi.org/10.1038/s41587-021-01180-3
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