In vivo RNA editing of point mutations via RNA-guided adenosine deaminases - PubMed (original) (raw)

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

Dhruva Katrekar et al. Nat Methods. 2019 Mar.

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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Conflict of interest statement

COMPETING FINANCIAL 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.

Figures

Figure 1:

Figure 1:. Engineering programmable RNA editing and characterizing specificity profiles:

(a) Schematics of RNA editing via constructs utilizing the full length ADAR2 and an engineered adRNA derived from the GluR2 transcript, or MS2 Coat Protein (MCP) fusions to the ADAR1/2 deaminase domains and the corresponding MS2 hairpin bearing adRNA. (b) Comparison of RNA editing efficiency of the endogenous RAB7A transcript by different RNA editing constructs quantified by Sanger sequencing (efficiency calculated as a ratio of Sanger peak heights G/(A+G)). Experiments were carried out in HEK 293T cells. Values represent mean +/− SEM (n=3). (c) Violin plots representing distributions of A->G editing yields observed at reference sites where at least one treatment sample was found to have a significant change (Fisher’s exact test, FDR = 1%) in editing yield relative to the control sample. Blue circles indicate editing yields at the target A-site within the RAB7A transcript. Black dots represent median off-target editing yields. To better visualize the shapes of the distributions, their maximum extent along the y-axis was equalized across all plots, and were truncated at 60% yield.

Figure 2:

Figure 2:. In vivo RNA editing in mouse models of human disease:

(a) Schematic of the DNA and RNA targeting approaches to restore dystrophin expression in the mdx mouse model of Duchenne Muscular Dystrophy: (i) a dual gRNA-CRISPR based approach leading to in frame excision of exon 23 and (ii) ADAR2 and MCP-ADAR1 based editing of the ochre codon. (b) Immunofluorescence staining for dystrophin in the TA muscle shows partial restoration of expression in treated samples (intra-muscular injections of AAV8-ADAR2, AAV8-ADAR2 (E488Q), and AAV8-CRISPR). Partial restoration of nNOS localization is also seen in treated samples (scale bar: 250μm). (c) In vivo TAA->TGG/TAG/TGA RNA editing efficiencies in corresponding treated adult mdx mice. Values represent mean +/− SEM (n=4, 3, 7, 3, 3, 10, 3, 4 independent TA muscles respectively). (d) Schematic of the OTC locus in the spf ash mouse model of Ornithine Transcarbamylase deficiency which have a G->A point mutation at a donor splice site in the last nucleotide of exon 4, and approach for correction of mutant OTC mRNA via ADAR2 mediated RNA editing. (e) In vivo RNA correction efficiencies in the correctly spliced OTC mRNA in the livers of treated adult spf ash mice (retro-orbital injections of AAV8-ADAR2 and AAV8-ADAR2 (E488Q)). Values represent mean +/− SEM (n=4, 4, 3, 3, 4, 5 independent animals respectively).

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