In vivo RNA editing of point mutations via RNA-guided adenosine deaminases

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Abstract

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

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Fig. 1: Engineering programmable RNA editing and characterizing specificity profiles.
Fig. 2: In vivo RNA editing in mouse models of human disease.

Data availability

Data are accessible via the NCBI GEO under accession GSE123905, and also are available from the corresponding author upon reasonable request. Source data for Figs. 1 and 2 and for Supplementary Figs. 16 and 1013 are available online.

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Acknowledgements

We thank A. Moreno, L. Hodge, D. Zhao, U. Parekh and other members of the Mali laboratory for advice and help with experiments, and the Salk GT3 viral core for help with AAV production. We also thank the UCSD IGM Genomics Center for providing NGS technologies and services. This work was generously supported by the Burroughs Wellcome Fund (no. 1013926 to P.M.) and the National Institutes of Health (grant numbers R01HG009285, RO1CA222826, and RO1GM123313 to P.M., and F32DK112682 to D.M.).

Author information

D.K. and P.M. conceived the study and wrote the paper. D.M. performed computational analyses and wrote the paper. D.K., G.C., A.G., A.W. and P.M. performed experiments. Y.S. and S.V. provided technical advice.

Correspondence to Prashant Mali.

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Competing interests

D.K. and P.M. have filed patents based on this work. P.M. is a scientific co-founder of Navega Therapeutics, Pretzel Therapeutics, Engine Biosciences and Shape Therapeutics. The terms of these arrangements have been reviewed and approved by the University of California, San Diego, in accordance with its conflict-of-interest policies.

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Integrated supplementary information

Supplementary Figure 1 Engineering GluR2 adRNAs: scaffold domain engineering.

Sequence information of adRNA scaffolds: ADAR recruiting domain, antisense RNA targeting domain and the cytosine mismatch are highlighted. Base pairs mutated to create stabilized scaffolds are numbered and highlighted in red, and the editing inducer element motif is shown in green. Quantification of editing efficiency of thus-generated scaffolds for the OTC reporter transcript quantified by Sanger sequencing is shown. Values represent mean ± s.e.m. (n = 3). All experiments were carried out in HEK293T cells. Source Data

Supplementary Figure 2 Engineering GluR2 adRNAs: antisense domain engineering.

a, Optimization of adRNA antisense region using adRNA scaffold 2: length and distance from the ADAR2 recruiting region were systematically varied. Values represent mean ± s.e.m. (n = 3). b, U6 promoter transcribed adRNAs with progressively longer antisense domain lengths, in combination with zero, one or two GluR2 domains, were evaluated for their ability to induce targeted RNA editing with or without exogenous ADAR2 expression. Values represent mean ± s.e.m. (n = 3). A portion of these data is reused in Fig. 1b. All the above experiments were carried out in HEK293T cells. c, Experimental confirmation of expression of endogenous ADAR1 and ADAR2 (relative to GAPDH) in HEK293T and HeLa cell lines. Observed levels were similar to those documented in The Human Protein Atlas (https://www.proteinatlas.org). Source Data

Supplementary Figure 3 Engineering MS2 adRNAs.

a, Systematic evaluation of antisense RNA targeting domain of the MS2 adRNA. Values represent mean ± s.e.m. (n = 3). b, On-target RNA editing by MCP–ADAR2 DD-NLS requires co-expression of the MS2 adRNA. GluR2 adRNA and MS2 adRNA used in this experiment had an antisense domain of length 20. Values represent mean ± s.e.m. (n = 3). All experiments were carried out in HEK293T cells. Source Data

Supplementary Figure 4 Analysis of RNA editing yields with U6 transcribed and chemically synthesized adRNAs.

a, Comparison of RNA editing efficiency of the OTC reporter transcript by GluR2 adRNA and MS2 adRNA guided RNA editing constructs, as well as the Cas13b-based REPAIR construct. Values represent mean ± s.e.m. (n = 6 for reporter and Cas13b-based constructs; n = 3 for all other constructs). b, Chemically synthesized adRNA versions were tested against a panel of mRNAs with or without exogenous ADAR2 expression. The exact chemical modifications are stated in the figure, along with the source of adRNA. Values represent mean ± s.e.m. (n = 3). All experiments were carried out in HEK293T cells. Source Data

Supplementary Figure 5 Analysis of RNA editing yields across a spectrum of endogenous targets chosen to cover a range of expression levels.

ADAR2- and MCP–ADAR-based editing efficiencies were compared across several endogenous transcripts. U6-transcribed long adRNAs with no or two GluR2 domains were also evaluated against multiple endogenous mRNA targets with or without exogenous ADAR2 expression. Values represent mean ± s.e.m. (n = 3). All experiments were carried out in HEK293T cells. Source Data

Supplementary Figure 6 ADAR2 variants and their effects on editing and specificity.

a, Comparison of on-target RNA editing and editing in flanking adenosines of the RAB7A transcript by GluR2 adRNA and MS2 adRNA guided RNA editing constructs, as well as the Cas13b-based REPAIR construct. Mean (n = 3) editing yields are depicted. b,c, ADAR2 (E488Q) exhibits higher efficiency than the ADAR2 in the in vitro editing of the spfash OTC reporter transcript (b; P = 0.037, unpaired t-test, two-tailed); values represent mean ± s.e.m. (n = 3) and mdx DMD reporter transcript (c; P = 0.048 and P = 0.012, respectively; unpaired t-test, two-tailed); values represent mean ± s.e.m. (n = 3). d, Comparison of the editing efficiency and specificity profiles of the ADAR2, ADAR2 (E488Q) and ADAR2 (∆1-138) for the OTC reporter transcript (upper panel) and endogenous RAB7A transcript (lower panel). Heat map indicates the A>G edits in the vicinity of the target (red arrow). Values represent mean ± s.e.m. (n = 3). All experiments were carried out in HEK293T cells, and editing efficiency was calculated as a ratio of Sanger peak heights G/(A + G). Source Data

Supplementary Figure 7 Transcriptome scale specificity profiles of RNA editing approaches.

2D histograms comparing the transcriptome-wide A>G editing yields observed with each construct (y-axis) to the yields observed with the control sample (x-axis). Each histogram represents the same set of 8,729,464 reference sites, where read coverage was at least 10 and at least one putative editing event was detected in at least one sample. Bins highlighted in red contain sites with significant changes in A>G editing yields when comparing treatment to control sample. Red crosses in each plot indicate the 100 sites with the smallest adjusted P values. Blue circles indicate the intended target A site within the RAB7A transcript. Large counts in bins near the lower-left corner likely correspond not only to low editing yields in both test and control samples, but also to sequencing errors and alignment errors. Large counts in bins near the upper-right corner of each plot likely correspond to homozygous single-nucleotide polymorphisms (SNPs), as well as other differences between the reference genome and the genome of the HEK293T cell line used in the experiments. GluR2 adRNA used in these experiments was the GluR2 adRNA(1,20,6).

Supplementary Figure 8 Variation of transcriptome scale editing specificity with construct features.

Each point in the box plots corresponds to the fraction of edited sites for one of the MCP–ADAR constructs listed in Fig. 1. We calculated the fraction of edited sites for each construct by dividing the number of reference sites with significant changes in A-to-G editing yield (see Supplementary Table 4) by 8,729,464, the total number of reference sites considered (Methods). Construct features indicated on the horizontal axes were compared using the Mann–Whitney U test, yielding P values of 0.16 for NLS versus NES, 0.0070 for ADAR1 versus ADAR2, 0.72 for “– adRNA” versus “+ adRNA,” and 0.038 for “ADAR WT” versus “ADAR E>Q” (n = 8 for all conditions).

Supplementary Figure 9 Transcriptome scale specificity profiles of RNA editing via long adRNAs.

2D histograms comparing the transcriptome-wide A>G editing yields observed with each construct (y-axis) to the yields observed with the control sample (x-axis). More details are provided in Supplementary Fig. 7.

Supplementary Figure 10 Optimization and evaluation of dystrophin RNA editing experiments in vitro and in vivo in mdx mice.

a, Schematic of RNA editing utilizing the full-length ADAR2 along with an engineered adRNA or a reverse-oriented adRNA (radRNA); (ii) RNA editing efficiencies of amber and ochre stop codons, in one step and two steps. Experiments were carried out in HEK293T cells. Values represent mean ± s.e.m. (n = 3). b, RNA editing of ochre codons requires two cytosine mismatches in the antisense RNA targeting domains of adRNA or radRNA to restore GFP expression. Experiments were carried out in HEK293T cells. Values represent mean ± s.e.m. (n = 3). c, Schematic of the AAV vectors utilized for in vivo delivery of adRNA and ADAR2, and in vitro optimization of RNA editing of amber and ochre stop codons in the presence of one or two copies of the adRNA, delivered via an AAV vector (P = 0.0003, P = 0.0001, P = 0.0015, respectively; unpaired t-test, two-tailed). Experiments were carried out in HEK293T cells. Values represent mean ± s.e.m. (n = 3 for reporters; n = 6 for all other conditions). d, Representative Sanger sequencing plot showing editing of the ochre stop codon (TAA>TGG) in the mdx DMD reporter transcript (quantified by NGS). Experiments were carried out in HEK293T cells (n = 3). e, Representative example of in vivo RNA editing analyses of treated mdx mice (quantified using NGS). Source Data

Supplementary Figure 11 Immunofluorescence and western blotting analyses of in vivo dystrophin RNA editing experiments in mdx mice.

a, Immunofluorescence staining for dystrophin in the TA muscle shows partial restoration of expression in treated samples (intramuscular injections of AAV8–ADAR2, AAV8–ADAR2 (E488Q), AAV8–MCP–ADAR1 (E1008Q) NLS). Partial restoration of nNOS is localization also seen in treated samples (scale bar, 250 μm). b, Western blots showing partial recovery of dystrophin expression (1–2.5%) in TA muscles of mdx mice injected with both components of the editing machinery, the enzyme and adRNA, and stable ADAR2 expression in injected TA muscles up to 8 weeks post-injection. c, Western blot showing partial restoration of dystrophin expression (10%) using AAV8–CRISPR. Source Data

Supplementary Figure 12 Optimization and evaluation of OTC RNA editing experiments in vitro and in vivo in spfash mice.

a, Representative Sanger sequencing plot showing correction of the point mutation in the spfash OTC reporter transcript (quantified using NGS). Experiments were carried out in HEK293T cells (n = 3). b, Representative example of in vivo RNA editing analyses of treated spfash mice showing correction of the point mutation in the correctly spliced OTC mRNA (quantified using NGS). c, In vivo RNA correction efficiencies in the OTC pre-mRNA in the livers of treated adult spfash mice (retro-orbital injections of AAV8–ADAR2 and AAV8–ADAR2 (E488Q). Values represent mean ± s.e.m. (n = 4, 4, 3, 3, 4, and 5 independent animals, respectively). d, PCR products showing the correctly and incorrectly spliced OTC mRNA. The incorrectly spliced mRNA is elongated by 48 bp. The fraction of incorrectly spliced mRNA is reduced in mice treated with adRNA + ADAR2 (E488Q). e, Western blot for OTC shows partial restoration (2.5–5%) of expression in treated adult spfash mice and stable ADAR2 (E488Q) expression 3 weeks post-injection. Source Data

Supplementary Figure 13 Assaying the effects of long adRNAs on target mRNA expression.

The effect of U6-transcribed long adRNAs on target mRNA expression level was evaluated against multiple endogenous mRNA targets (P = 0.7431, P = 0.1015, P = 0.3671, P = 0.2086, P = 0.0026, P = 0.0676, unpaired t-test, two-tailed); values represent mean ± s.e.m. (n = 3 or 4). Experiments were carried out in HEK293T cells. Source Data

Supplementary Figure 14 Toxicity analyses of in vivo RNA editing experiments.

Summary of animal experiments documenting the route of AAV administration, construct delivered, and health of injected mice 3 weeks post-injection.

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Katrekar, D., Chen, G., Meluzzi, D. et al. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat Methods 16, 239–242 (2019) doi:10.1038/s41592-019-0323-0

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