Applying Human ADAR1p110 and ADAR1p150 for Site-Directed RNA Editing—G/C Substitution Stabilizes GuideRNAs against Editing

Heep, Madeleine; Mach, Pia; Reautschnig, Philipp; Wettengel, Jacqueline; Stafforst, Thorsten

doi:10.3390/genes8010034

Cited by 27 publications

(21 citation statements)

References 28 publications

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“… 95 However, attempts are on the way to also retarget the ubiquitously expressed ADAR1 which should eliminate the need for enzyme expression and would only require expression or delivery of a well designed guide RNA. 96 …”

Section: A-i Editing In Mammalsmentioning

confidence: 99%

A to I editing in disease is not fake news

et al. 2017

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Adenosine deaminases acting on RNA (ADARs) are zinc-containing enzymes that deaminate adenosine bases to inosines within dsRNA regions in transcripts. In short, structured dsRNA hairpins individual adenosine bases may be targeted specifically and edited with up to one hundred percent efficiency, leading to the production of alternative protein variants. However, the majority of editing events occur within longer stretches of dsRNA formed by pairing of repetitive sequences. Here, many different adenosine bases are potential targets but editing efficiency is usually much lower.Recent work shows that ADAR-mediated RNA editing is also required to prevent aberrant activation of antiviral innate immune sensors that detect viral dsRNA in the cytoplasm. Missense mutations in the ADAR1 RNA editing enzyme cause a fatal auto-inflammatory disease, Aicardi–Goutières syndrome (AGS) in affected children. In addition RNA editing by ADARs has been observed to increase in many cancers and also can contribute to vascular disease. Thus the role of RNA editing in the progression of various diseases can no longer be ignored.The ability of ADARs to alter the sequence of RNAs has also been used to artificially target model RNAs in vitro and in cells for RNA editing. Potentially this approach may be used to repair genetic defects and to alter genetic information at the RNA level.In this review we focus on the role of ADARs in disease development and progression and on their potential use to artificially modify RNAs in a targeted manner.

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Section: A-i Editing In Mammalsmentioning

confidence: 99%

A to I editing in disease is not fake news

et al. 2017

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“…9,10,12-17 SDRE has successfully been employed in vitro, in mammalian cells, and in a simple animal model. [9][10][11][12][13][14][15][16][17] It has been used to target a variety of genetic mutations that lead to diseases, including Cystic Fibrosis, 10 Duchenne muscular dystrophy 8 and Factor V Leiden thrombophilia. 13 Targeting ADAR activity has also been proven useful for basic research.…”

Section: Introductionmentioning

confidence: 99%

“…Others have reported few or no off-target edits, yet they have only looked for them in the targeted message. 9,[11][12][13][14][15]17 Our group uncovered numerous off-target events within the targeted message, and they could be mostly, but not completely, eliminated by reducing the amount of RNA guide transfected. 16 We also explored off-target edits in five endogenous mRNAs expressed in HEK293T cells that were not targeted by our RNA guide and saw no editing.…”

Section: Introductionmentioning

confidence: 99%

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme

et al. 2017

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Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

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“…In the constant attempt to reduce the off-target effects of RNA editing, several groups proposed the idea of taking advantage of the naturally occurring ADARs. Extending the idea of the early study of Woolf where the possibility of recruiting an endogenous ADAR in Xenopus was proved [36], recent studies developed principle-based similar approaches [37][38][39]. The first study of such manner in a human study models, managed to modify in Hela cells a mutation within PINK1 associated with loss of function effects and forced PINK1 connected mitophagy; this was done by designing a gRNA capable to capture human ADAR2 toward selected mRNA sites [37].…”

Section: Is Rna-based Genetic Engineering the Safer Option?mentioning

confidence: 99%

CRISPR-based RNA editing: diagnostic applications and therapeutic options

Gulei

Ráduly

Berindan‐Neagoe

et al. 2019

Expert Review of Molecular Diagnostics

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Gene therapy is currently in the front rows of biomedical research with an increased focus on direct nuclease-based editing. If until now the research was mainly concentrated on modifications at the DNA level, new adapted tolls are starting to gain momentum due to their ability to induce changes within the RNA sequence. The advantage consists in the half-life of the messenger RNA and implicit the non-permanent character of the editing strategy. Recently, the ability of nucleases like Cas12a and Cas13 to cleave DNA and RNA, respectively, was used for detection of low particle infectious agents with high sensitivitye.g. DETECTOR and SHERLOCK Systems. The present article briefly presents the latest advancements from the gene editing therapeutic area, as well as the new diagnostic tools with great likelihoods within the clinical environment.

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Applying Human ADAR1p110 and ADAR1p150 for Site-Directed RNA Editing—G/C Substitution Stabilizes GuideRNAs against Editing

Cited by 27 publications

References 28 publications

A to I editing in disease is not fake news

A to I editing in disease is not fake news

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme

CRISPR-based RNA editing: diagnostic applications and therapeutic options

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