RGEN Editing of RNA and DNA: The Long and Winding Road from Catalytic RNAs to CRISPR to the Clinic.

The first clinical studies utilizing RNA-guided endonucleases (RGENs) to therapeutically edit RNA and DNA in cancer patients were recently published. These groundbreaking technological advances promise to revolutionize genetic therapy and, as I discuss, represent the culmination of decades of innovative work to engineer RGENs for such editing applications.

CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.

Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.

RNA Pol II-dependent transcription efficiency fine-tunes A-to-I editing levels.

A-to-I RNA editing is a widespread epitranscriptomic phenomenon leading to the conversion of adenosines to inosines, which are primarily interpreted as guanosines by cellular machines. Consequently, A-to-I editing can alter splicing or lead to recoding of transcripts. As misregulation of editing can cause a variety of human diseases, A-to-I editing requires tight regulation of the extent of deamination, particularly in protein-coding regions. The bulk of A-to-I editing occurs cotranscriptionally. Thus, we studied A-to-I editing regulation in the context of transcription and pre-mRNA processing. We show that stimulation of transcription impacts editing levels. Activation of the transcription factor MYC leads to an up-regulation of A-to-I editing, particularly in transcripts that are suppressed upon MYC activation. Moreover, low pre-mRNA synthesis rates and low pre-mRNA expression levels support high levels of editing. We also show that editing levels greatly differ between nascent pre-mRNA and mRNA in a cellular system, as well as in mouse tissues. Editing levels can increase or decrease from pre-mRNA to mRNA and can vary across editing targets and across tissues, showing that pre-mRNA processing is an important layer of editing regulation. Several lines of evidence suggest that the differences emerge during pre-mRNA splicing. Moreover, actinomycin D treatment of primary neuronal cells and editing level analysis suggests that regulation of editing levels also depends on transcription.

Isoforms of RNA-Editing Enzyme ADAR1 Independently Control Nucleic Acid Sensor MDA5-Driven Autoimmunity and Multi-organ Development.

Mutations in ADAR, which encodes the ADAR1 RNA-editing enzyme, cause Aicardi-Goutières syndrome (AGS), a severe autoimmune disease associated with an aberrant type I interferon response. How ADAR1 prevents autoimmunity remains incompletely defined. Here, we demonstrate that ADAR1 is a specific and essential negative regulator of the MDA5-MAVS RNA sensing pathway. Moreover, we uncovered a MDA5-MAVS-independent function for ADAR1 in the development of multiple organs. We showed that the p150 isoform of ADAR1 uniquely regulated the MDA5 pathway, whereas both the p150 and p110 isoforms contributed to development. Abrupt deletion of ADAR1 in adult mice revealed that both of these functions were required throughout life. Our findings delineate genetically separable roles for both ADAR1 isoforms in vivo, with implications for the human diseases caused by ADAR mutations.

Extensive C->U transition biases in the genomes of a wide range of mammalian RNA viruses; potential associations with transcriptional mutations, damage- or host-mediated editing of viral RNA.

The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, proposed to be driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 36 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x-7.5x), with a consistently observed favoured 5' U upstream context. The presence of genome scale RNA secondary structure (GORS) was the only other genomic or structural parameter significantly associated with C->U/U->C transition asymmetries by multivariable analysis (ANOVA), potentially reflecting RNA structure dependence of sites targeted for C->U mutations. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, potentially paralleling extensive homoplasy of this transition reported in SARS-CoV-2. Although mechanisms remain to be functionally characterised, excess C->U substitutions accounted for 11-14% of standing sequence variability of structured viruses and may therefore represent a potent driver of their sequence diversification and longer-term evolution.

RNA editing alterations define manifestation of prion diseases.

Prion diseases are fatal neurodegenerative disorders caused by misfolding of the normal prion protein into an infectious cellular pathogen. Clinically characterized by rapidly progressive dementia and accounting for 85% of human prion disease cases, sporadic Creutzfeldt-Jakob disease (sCJD) is the prevalent human prion disease. Although sCJD neuropathological hallmarks are well-known, associated molecular alterations are elusive due to rapid progression and absence of preclinical stages. To investigate transcriptome alterations during disease progression, we utilized tg340-PRNP129MM mice infected with postmortem material from sCJD patients of the most susceptible genotype (MM1 subtype), a sCJD model that faithfully recapitulates the molecular and pathological alterations of the human disease. Here we report that transcriptomic analyses from brain cortex in the context of disease progression, reveal epitranscriptomic alterations (specifically altered RNA edited pathway profiles, eg., ER stress, lysosome) that are characteristic and possibly protective mainly for preclinical and clinical disease stages. Our results implicate regulatory epitranscriptomic mechanisms in prion disease neuropathogenesis, whereby RNA-editing targets in a humanized sCJD mouse model were confirmed in pathological human autopsy material.

RNA editing-mediated regulation of calcium-dependent activator protein for secretion (CAPS1) localization and its impact on synaptic transmission.

Calcium-dependent activator protein for secretion 1 (CAPS1) is a SNARE accessory protein that facilitates formation of the SNARE complex to enable neurotransmitter release. Messenger RNAs encoding CAPS1 are subject to a site-specific adenosine-to-inosine (A-to-I) editing event resulting in a glutamate-to-glycine (E-to-G) substitution in the C-terminal domain of the encoded protein product. The C-terminal domain of CAPS1 is necessary for its synaptic enrichment and Cadps RNA editing has been shown previously to enhance the release of neuromodulatory transmitters. Using mutant mouse lines engineered to solely express CAPS1 protein isoforms encoded by either the non-edited or edited Cadps transcript, primary neuronal cultures from mouse hippocampus were used to explore the effect of Cadps editing on neurotransmission and CAPS1 synaptic localization at both glutamatergic and GABAergic synapses. While the editing of Cadps does not alter baseline evoked neurotransmission, it enhances short-term synaptic plasticity, specifically short-term depression, at inhibitory synapses. Cadps editing also alters spontaneous inhibitory neurotransmission. Neurons that solely express edited Cadps have a greater proportion of synapses that contain CAPS1 than neurons that solely express non-edited Cadps for both glutamatergic and GABAergic synapses. Editing of Cadps transcripts is regulated by neuronal activity, as global network stimulation increases the extent of transcripts edited in wild-type hippocampal neurons, whereas chronic network silencing decreases the level of Cadps editing. Taken together, these results provide key insights into the importance of Cadps editing in modulating its own synaptic localization, as well as the modulation of neurotransmission at inhibitory synapses in hippocampal neurons.

The landscape of miRNA editing in animals and its impact on miRNA biogenesis and targeting.

Adenosine-to-inosine (A-to-I) RNA editing regulates miRNA biogenesis and function. To date, fewer than 160 miRNA editing sites have been identified. Here, we present a quantitative atlas of miRNA A-to-I editing through the profiling of 201 pri-miRNA samples and 4694 mature miRNA samples in human, mouse, and Drosophila. We identified 4162 sites present in ∼80% of the pri-miRNAs and 574 sites in mature miRNAs. miRNA editing is prevalent in many tissue types in human. However, high-level editing is mostly found in neuronal tissues in mouse and Drosophila Interestingly, the edited miRNAs in neuronal and non-neuronal tissues in human gain two distinct sets of new targets, which are significantly associated with cognitive and organ developmental functions, respectively. Furthermore, we reveal that miRNA editing profoundly affects asymmetric strand selection. Altogether, these data provide insight into the impact of RNA editing on miRNA biology and suggest that miRNA editing has recently gained non-neuronal functions in human.

Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs.

Molecular tools that target RNA at specific sites allow recoding of RNA information and processing. SNAP-tagged deaminases guided by a chemically stabilized guide RNA can edit targeted adenosine to inosine in several endogenous transcripts simultaneously, with high efficiency (up to 90%), high potency, sufficient editing duration, and high precision. We used adenosine deaminases acting on RNA (ADARs) fused to SNAP-tag for the efficient and concurrent editing of two disease-relevant signaling transcripts, KRAS and STAT1. We also demonstrate improved performance compared with that of the recently described Cas13b-ADAR.

Deciphering the Biological Significance of ADAR1-Z-RNA Interactions.

Adenosine deaminase acting on RNA 1 (ADAR1) is an enzyme responsible for double-stranded RNA (dsRNA)-specific adenosine-to-inosine RNA editing, which is estimated to occur at over 100 million sites in humans. ADAR1 is composed of two isoforms transcribed from different promoters: p150 and N-terminal truncated p110. Deletion of ADAR1 p150 in mice activates melanoma differentiation-associated protein 5 (MDA5)-sensing pathway, which recognizes endogenous unedited RNA as non-self. In contrast, we have recently demonstrated that ADAR1 p110-mediated RNA editing does not contribute to this function, implying that a unique Z-DNA/RNA-binding domain α (Zα) in the N terminus of ADAR1 p150 provides specific RNA editing, which is critical for preventing MDA5 activation. In addition, a mutation in the Zα domain is identified in patients with Aicardi-Goutières syndrome (AGS), an inherited encephalopathy characterized by overproduction of type I interferon. Accordingly, we and other groups have recently demonstrated that Adar1 Zα-mutated mice show MDA5-dependent type I interferon responses. Furthermore, one such mutant mouse carrying a W197A point mutation in the Zα domain, which inhibits Z-RNA binding, manifests AGS-like encephalopathy. These findings collectively suggest that Z-RNA binding by ADAR1 p150 is essential for proper RNA editing at certain sites, preventing aberrant MDA5 activation.

RNA editing in bacteria recodes multiple proteins and regulates an evolutionarily conserved toxin-antitoxin system.

Adenosine (A) to inosine (I) RNA editing is widespread in eukaryotes. In prokaryotes, however, A-to-I RNA editing was only reported to occur in tRNAs but not in protein-coding genes. By comparing DNA and RNA sequences of Escherichia coli, we show for the first time that A-to-I editing occurs also in prokaryotic mRNAs and has the potential to affect the translated proteins and cell physiology. We found 15 novel A-to-I editing events, of which 12 occurred within known protein-coding genes where they always recode a tyrosine (TAC) into a cysteine (TGC) codon. Furthermore, we identified the tRNA-specific adenosine deaminase (tadA) as the editing enzyme of all these editing sites, thus making it the first identified RNA editing enzyme that modifies both tRNAs and mRNAs. Interestingly, several of the editing targets are self-killing toxins that belong to evolutionarily conserved toxin-antitoxin pairs. We focused on hokB, a toxin that confers antibiotic tolerance by growth inhibition, as it demonstrated the highest level of such mRNA editing. We identified a correlated mutation pattern between the edited and a DNA hard-coded Cys residue positions in the toxin and demonstrated that RNA editing occurs in hokB in two additional bacterial species. Thus, not only the toxin is evolutionarily conserved but also the editing itself within the toxin is. Finally, we found that RNA editing in hokB increases as a function of cell density and enhances its toxicity. Our work thus demonstrates the occurrence, regulation, and functional consequences of RNA editing in bacteria.

Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria.

RNA editing is converting hundreds of cytosines into uridines during organelle gene expression of land plants. The pentatricopeptide repeat (PPR) proteins are at the core of this posttranscriptional RNA modification. Even if a PPR protein defines the editing site, a DYW domain of the same or another PPR protein is believed to catalyze the deamination. To give insight into the organelle RNA editosome, we performed tandem affinity purification of the plastidial CHLOROPLAST BIOGENESIS 19 (CLB19) PPR editing factor. Two PPR proteins, dually targeted to mitochondria and chloroplasts, were identified as potential partners of CLB19. These two proteins, a P-type PPR and a member of a small PPR-DYW subfamily, were shown to interact in yeast. Insertional mutations resulted in embryo lethality that could be rescued by embryo-specific complementation. A transcriptome analysis of these complemented plants showed major editing defects in both organelles with a very high PPR type specificity, indicating that the two proteins are core members of E+-type PPR editosomes.

The evolution and adaptation of A-to-I RNA editing.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification that affects the information encoded from DNA to RNA to protein. RNA editing can generate a multitude of transcript isoforms and can potentially be used to optimize protein function in response to varying conditions. In light of this and the fact that millions of editing sites have been identified in many different species, it is interesting to examine the extent to which these sites have evolved to be functionally important. In this review, we discuss results pertaining to the evolution of RNA editing, specifically in humans, cephalopods, and Drosophila. We focus on how comparative genomics approaches have aided in the identification of sites that are likely to be advantageous. The use of RNA editing as a mechanism to adapt to varying environmental conditions will also be reviewed.

Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids.

Recent identification of several different types of RNA editing factors in plant organelles suggests complex RNA editosomes within which each factor has a different task. However, the precise protein interactions between the different editing factors are still poorly understood. In this paper, we show that the E+-type pentatricopeptide repeat (PPR) protein SLO2, which lacks a C-terminal cytidine deaminase-like DYW domain, interacts in vivo with the DYW-type PPR protein DYW2 and the P-type PPR protein NUWA in mitochondria, and that the latter enhances the interaction of the former ones. These results may reflect a protein scaffold or complex stabilization role of NUWA between E+-type PPR and DYW2 proteins. Interestingly, DYW2 and NUWA also interact in chloroplasts, and DYW2-GFP overexpressing lines show broad editing defects in both organelles, with predominant specificity for sites edited by E+-type PPR proteins. The latter suggests a coordinated regulation of organellar multiple site editing through DYW2, which probably provides the deaminase activity to E+ editosomes.

RNA editing blood biomarkers for predicting mood alterations in HCV patients.

Treatment-emergent depression is a common complication in patients with chronic hepatitis C virus (HCV) infection undergoing antiviral combination therapy with IFN-α and ribavirin. It has recently been shown that changes in A-to-I RNA editing rates are associated with various pathologies such as inflammatory disorders, depression and suicide. Interestingly, IFN-α induces gene expression of the RNA editing enzyme ADAR1-1 (ADAR1a-p150) and alters overall RNA editing activity. In this study, we took advantage of the high prevalence of pharmacologically induced depression in patients treated with IFN-α and ribavirin to test the interest of RNA editing-related biomarkers in white blood cells of patients. In this 16-week longitudinal study, a small cohort of patients was clinically evaluated using standard assessment methods prior to and during antiviral therapy and blood samples were collected to analyse RNA editing modifications. A-I RNA editing activity on the phosphodiesterase 8A (PDE8A) gene, a previously identified RNA editing hotspot in the context of lupus erythematosus, was quantified by using an ultra-deep next-generation sequencing approach. We also monitored gene expression levels of the ADAR enzymes and the PDE8A gene during treatment by qPCR. As expected, psychiatric evaluation could track treatment-emergent depression, which occurred in 30% of HCV patients. We show that PDE8A RNA editing is increased in all patients following interferon treatment, but differently in 30% of patients. This effect was mimicked in a cellular model using SHSY-5Y neuroblastoma cells. By combining the data of A-I RNA editing and gene expression, we generated an algorithm that allowed discrimination between the group of patients who developed a treatment-emergent depression and those who did not. The current model of drug-induced depression identified A-I RNA editing biomarkers as useful tools for the identification of individuals at risk of developing depression in an objective, quantifiable biological blood test.

Comparative RNA editing profile of mitochondrial transcripts in cytoplasmic male sterile and fertile pigeonpea reveal significant changes at the protein level.

RNA editing is a process which leads to post-transcriptional alteration of the nucleotide sequence of the corresponding mRNA molecule which may or may not lead to changes at the protein level. Apart from its role in providing variability at the transcript and protein levels, sometimes, such changes may lead to abnormal expression of the mitochondrial gene leading to a cytoplasmic male sterile phenotype. Here we report the editing status of 20 major mitochondrial transcripts in both male sterile (AKCMS11) and male fertile (AKPR303) pigeonpea genotypes. The validation of the predicted editing sites was done by mapping RNA-seq reads onto the amplified mitochondrial genes, and 165 and 159 editing sites were observed in bud tissues of the male sterile and fertile plant respectively. Among the resulting amino acid alterations, the most frequent one was the conversion of hydrophilic amino acids to hydrophobic. The alterations thus detected in our study indicates differential editing, but no major change in terms of the abnormal protein structure was detected. However, the above investigation provides an insight into the behaviour of pigeonpea mitochondrial genome in native and alloplasmic state and could hold clues in identification of editing factors and their role in adaptive evolution in pigeonpea.

RNA Editing by ADAR Adenosine Deaminases: From Molecular Plasticity of Neural Proteins to the Mechanisms of Human Cancer.

RNA editing by adenosine deaminases of the ADAR family attracts a growing interest of researchers, both zoologists studying ecological and evolutionary plasticity of invertebrates and medical biochemists focusing on the mechanisms of cancer and other human diseases. These enzymes deaminate adenosine residues in the double-stranded (ds) regions of RNA with the formation of inosine. As a result, some RNAs change their three-dimensional structure and functions. Adenosine-to-inosine editing in the mRNA coding sequences may cause amino acid substitutions in the encoded proteins. Here, we reviewed current concepts on the functions of two active ADAR isoforms identified in mammals (including humans). The ADAR1 protein, which acts non-specifically on extended dsRNA regions, is capable of immunosuppression via inactivation of the dsRNA interactions with specific sensors inducing the cell immunity. Expression of a specific ADAR1 splicing variant is regulated by the type I interferons by the negative feedback mechanism. It was shown that immunosuppressing effects of ADAR1 facilitate progression of some types of cancer. On the other hand, changes in the amino acid sequences resulting from the mRNA editing by the ADAR enzymes can result in the formation of neoantigens that can activate the antitumor immunity. The ADAR2 isoform acts on RNA more selectively; its function is associated with the editing of mRNA coding regions and can lead to the amino acid substitutions, in particular, those essential for the proper functioning of some neurotransmitter receptors in the central nervous system.

MORF9 Functions in Plastid RNA Editing with Tissue Specificity.

RNA editing in plant mitochondria and plastids converts specific nucleotides from cytidine (C) to uridine (U). These editing events differ among plant species and are relevant to developmental stages or are impacted by environmental conditions. Proteins of the MORF family are essential components of plant editosomes. One of the members, MORF9, is considered the core protein of the editing complex and is involved in the editing of most sites in chloroplasts. In this study, the phenotypes of a T-DNA insertion line with loss of MORF9 and of the genetic complementation line of Arabidopsis were analyzed, and the editing efficiencies of plastid RNAs in roots, rosette leaves, and flowers from the morf9 mutant and the wild-type (WT) control were compared by bulk-cDNA sequencing. The results showed that most of the known MORF9-associated plastid RNA editing events in rosette leaves and flowers were similarly reduced by morf9 mutation, with the exception that the editing rate of the sites ndhB-872 and psbF-65 declined in the leaves and that of ndhB-586 decreased only in the flowers. In the roots, however, the loss of MORF9 had a much lower effect on overall plastid RNA editing, with nine sites showing no significant editing efficiency change, including accD-794, ndhD-383, psbZ-50, ndhF-290, ndhD-878, matK-706, clpP1-559, rpoA-200, and ndhD-674, which were reduced in the other tissues. Furthermore, we found that during plant aging, MORF9 mRNA level, but not the protein level, was downregulated in senescent leaves. On the basis of these observations, we suggest that MORF9-mediated RNA editing is tissue-dependent and the resultant organelle proteomes are pertinent to the specific tissue functions.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

The RNA Editing Factor SlORRM4 Is Required for Normal Fruit Ripening in Tomato.

RNA editing plays a key posttranscriptional role in gene expression. Existing studies on cytidine-to-uridine RNA editing in plants have focused on maize (Zea mays), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana). However, the importance and regulation of RNA editing in several critical agronomic processes are not well understood, a notable example of which is fruit ripening. Here, we analyzed the expression profile of 33 RNA editing factors and identified 11 putative tomato (Solanum lycopersicum) fruit ripening-related factors. A rapid virus-induced gene silencing assay indicated that the organelle RNA recognition motif-containing protein SlORRM4 affected tomato fruit ripening. Knocking out SlORRM4 expression using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome editing strategy delayed tomato fruit ripening by lowering respiratory rate and ethylene production. Additionally, the expression of numerous genes associated with fruit ripening and mitochondrial functions changed significantly when SlORRM4 was knocked out. Moreover, the loss of SlORRM4 function significantly reduced RNA editing of many mitochondrial transcripts, leading to low-level expression of some core subunits that are critical for mitochondrial complex assembly (i.e. Nad3, Cytc1, and COX II). Taken together, these results indicate that SlORRM4 is involved in RNA editing of transcripts in ripening fruit that influence mitochondrial function and key aspects of fruit ripening.