Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs

Abstract

Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-l-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.

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Fig. 1: Leveraging endogenous ADAR1 protein for targeted RNA editing.
Fig. 2: Characterization and optimization of LEAPER.
Fig. 3: Editing endogenous transcripts with LEAPER.
Fig. 4: Transcriptome-wide specificity of RNA editing by LEAPER.
Fig. 5: RNA editing in multiple human primary cells by LEAPER.
Fig. 6: Restoration of transcriptional regulatory activity of mutant TP53W53X by LEAPER.
Fig. 7: Restoration of IDUA activity in Hurler syndrome patient-derived primary fibroblast by LEAPER.

Data availability

All data presented in this manuscript are available from the corresponding author upon reasonable request. Transcriptome-wide RNA-seq data are accessible via the NCBI Sequence Read Archive database with accession code PRJNA544353.

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Acknowledgements

We acknowledge the staff of the BIOPIC High-throughput Sequencing Center (Peking University) and Genetron Health for their assistance in NGS analysis, the National Center for Protein Sciences (Beijing) and the core facilities at the School of Life Sciences (Peking University, X. Zhang, F. Wang and L. Du) for help with Fluorescence Activated Cell Sorting. We thank the High-Performance Computing Platform at Peking University for providing platforms of NGS data analysis. We thank M. Mo for technical assistance, J. Wang for providing plasmids encoding disease-relevant genes and primary cells and we also thank Z. Jiang for providing the mouse melanoma cell line B16. This project was supported by funds from Beijing Municipal Science & Technology Commission (grant no. Z181100001318009), the National Science Foundation of China (no. 31430025), Beijing Advanced Innovation Center for Genomics at Peking University and the Peking-Tsinghua Center for Life Sciences (to W.W.); the National Science Foundation of China (no. 31870893) and the National Major Science & Technology Project for Control and Prevention of Major Infectious Diseases in China (no. 2018ZX10301401, to Z.Z.) and the Beijing Nova Program (no. Z181100006218042, to P.Y.).

Author information

W.W. conceived and supervised the project. W.W., L.Q., Z.Y., S.Z., C.W., Z.C. and Z.Z. designed the experiments. L.Q., Z.Y., C.W., S.Z., Z.C. and P.Y. performed the experiments with the help from F.T., Y.B. and Y.Z. Y.Y. conducted all the sample preparation for NGS. Z.Y. and Z.L. performed the data analysis. L.Q., S.Z., Z.Z. and W.W. wrote the manuscript with the help of all other authors.

Correspondence to Wensheng Wei.

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Competing interests

A patent has been filed relating to the data presented. W.W. is a founder andscientific adviser for EdiGene.

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Integrated supplementary information

Supplementary Figure 1 Exploration of an efficient RNA editing platform.

(a) Schematic of dLbuCas13a-ADAR1DD (E1008Q) fusion protein and the corresponding crRNA. The catalytic inactive LbuCas13a was fused to the deaminase domain of ADAR1 (hyperactive E1008Q variant) using 3× GGGGS linker. The crRNA (crRNACas13a) consisted of Lbu-crRNA scaffold and a spacer which was complementary to the targeting RNA with an A-C mismatch as indicated. (b) Schematic of dual fluorescent reporter system and the Lbu-crRNA with various lengths of spacers as indicated. (c) Quantification of the EGFP positive (EGFP+) cells. HEK293T cells stably expressing the Repoter-1 were transfected with indicated lengths of crRNACas13a, with or without co-expression of the dLbuCas13a-ADAR1DD (E1008Q), followed by FACS analysis. (d) Representative FACS result from the experiment performed with the Ctrl crRNACas13a (70-nt random spacer) or crRNACas13a (70-nt targeting spacer). (e) Representative FACS result from the experiment performed with REPAIR. Left, the Ctrl crRNACas13b (70-nt random spacer) or crRNACas13b (70-nt targeting spacer) was co-transfected with or without dPspCas13b-ADAR2DD-E488Q into HEK293T cells, which stably express Reporter-1. Right, the Ctrl crRNACas13b (70-nt random spacer) or crRNACas13b (70-nt targeting spacer) was co-transfected with Reporter-1 into HEK293T ADAR1−/− cells, in the presence or absence of dPspCas13b-ADAR2DD-E488Q. The 70-nt random or targeting spacer was fused to the 3′-end of PspCas13b-crRNA scaffold. The RNA editing effects were quantified by the percentages of EGFP+ cells. Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel).

Supplementary Figure 2 mRNA expression level of ADAR1/ADAR2 and arRNA-mediated RNA editing.

(a) Quantitative PCR showing the mRNA levels of ADAR1 and ADAR2 in HEK293T cells. Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel). (b) Representative FACS results from Fig. 1e.

Supplementary Figure 3 Quantitative PCR showing the effects of LEAPER on the expression levels of targeted Reporter-1 transcripts by 111-nt arRNA or control RNA in HEK293T cells.

Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel); unpaired two-sided Student’s t-test, ns, not significant.

Supplementary Figure 4 Targeted RNA editing with LEAPER in multiple cell lines.

(a) Western-blot results showing the expression levels of ADAR1, ADAR2 and ADAR3 in indicated human cell lines. β-tubulin was used as a loading control. Data shown is the representative of three independent experiments. ADAR1−/−/ADAR2 represents ADAR1-knockout HEK293T cells overexpressing ADAR2. (b) Relative ADAR protein expression levels normalized by β-tubulin expression. (c) Indicated human cells were transfected with Reporter-1, along with the 71-nt control arRNA (Ctrl RNA71) or with the 71-nt targeting arRNA (arRNA71) followed by FACS analysis. (d) Indicated mouse cell lines were analyzed as described in (c). EGFP+ percentages were normalized by transfection efficiency, which was determined by mCherry+. Error bars in (b, c, d) all indicate the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel); unpaired two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Supplementary Figure 5

Schematic of Reporter-1 (a), -2 (b), and -3 (c), as well as their corresponding arRNAs.

Supplementary Figure 6 Effects of LEAPER on the expression levels of targeted transcripts and protein products.

(a) Quantitative PCR showing the expression levels of targeted transcripts from PPIB, KRAS, SMAD4 and FANCC by the corresponding 151-nt arRNA or Control RNA in HEK293T cells. Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel); unpaired two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (b) Western blot results showing the effects on protein products of targeted KRAS gene by 151-nt arRNA in HEK293T cells. β-tubulin was used as a loading control.

Supplementary Figure 7 Editing endogenous transcripts with LEAPER.

(a) Schematic of the KARS transcript sequence covered by the 151-nt arRNA. The arrow indicates the targeting adenosine. All adenosines were marked in red. (b) Heatmap of editing rate on adenosines covered by indicated arRNAs in the KARS transcript (marked in the bold frame in blue). (c) Schematic of the SMAD4 transcript covered by the 151-nt arRNA. (d) Heatmap of editing rate on adenosines covered by indicated arRNAs in the SMAD4 transcript. (e) Schematic of the FANCC transcript covered by the 151-nt arRNA. (f) Heatmap of editing rate on adenosines covered by indicated arRNAs in the FANCC transcript. For each arRNA, the region of duplex RNA is highlighted with bold frame in blue. Data (b, d, and f) are presented as the mean (n = 3, n represents the number of independent experiments performed in parallel).

Supplementary Figure 8 Evaluation of potential off-targets.

(a) Top, schematic of the highly complementary region between arRNA151-PPIB and the indicated potential off-target sites, which were predicted by searching homologous sequences through NCBI-BLAST. Bottom, Deep sequencing showing the editing rate on the on-target site and all predicted off-target sites of arRNA151-PPIB. Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel). (b) Schematic of the highly complementary region of arRNA111-FANCC and the indicated potential off-target sequence, which were predicted by searching homologous sequences through NCBI-BLAST. (c) Deep sequencing showing the editing rate on the on-target site and all predicted off-target sites of arRNA111-FANCC. All data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel).

Supplementary Figure 9 Differential gene expression analysis with RNA-seq data at the transcriptome level.

Left, differential gene expression analysis between Ctrl RNA151 and Mock. Middle, differential gene expression analysis between arRNA151-PPIB and Mock. Right, differential gene expression analysis between arRNA151-PPIB and Ctrl RNA151. The DESeq2 (version 1.18.1) tool was used to analyze the differential gene expression with the FPKM expression data. Genes with adjusted P value smaller than 0.01 and log2(fold change) larger than 2 were viewed as significantly differentially expressed genes, labelled in red. Genes that were not differentially expressed were labelled in grey. The targeted gene PPIB was labelled in blue. Four independent experiments were performed.

Supplementary Figure 10 Safety evaluation of applying LEAPER in mammalian cells.

(a and b) Effect of arRNA transfection on innate immune response. The indicated arRNAs or the poly(I:C) were transfected into HEK293T cells. Total RNA was then analyzed using quantitative PCR to determine expression levels of IFN-β (a) and IL-6 (b). Data (a and b) are presented as the mean ±s.e.m. (n = 3, n represents the number of independent experiments performed in parallel).

Supplementary Figure 11 Correction of pathogenic mutations by LEAPER.

(a) Schematic representation of the selected disease-relevant cDNA containing G to A mutation from ClinVar data and the corresponding 111-nt arRNA. (b) A to I correction of disease-relevant G>A mutation from ClinVar data by the corresponding 111-nt arRNA, targeting clinical-related mutations from six pathogenic genes as indicated (Supplementary Tables 2 and 4). Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel); unpaired two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Supplementary Figure 12 Editing mutant TP53W53X transcripts by LEAPER.

Top, schematic of the TP53 transcript sequence covered by the 111-nt arRNAs. The arrow indicates the targeted adenosine. All adenosines were marked in red. Bottom, a heatmap of editing rate on adenosines covered by indicated arRNAs in the TP53 transcript. Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel).

Supplementary information

Supplementary Figs.

Supplementary Figs. 1–12

Reporting Summary

Supplementary Table 1

Cas13 crRNA sequences

Supplementary Table 2

Sequences of arRNAs and control RNAs used in this study

Supplementary Table 3

Differential gene expression results

Supplementary Table 4

Disease-relevant cDNAs used in this study

Supplementary Table 5

Primers used in this study

Supplementary Materials

Supplementary sequences

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