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1.
Methods ; 156: 16-24, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30502398

RESUMEN

Adenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A's) to inosines (I's) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I's base pair like guanosines (G's). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. This ability to re-code makes ADAR an attractive therapeutic tool to correct genetic mutations within mRNA. The main challenge in doing so is to re-direct ADAR's catalytic activity towards A's that are not naturally edited, a process termed Site-Directed RNA Editing (SDRE). Recently, a handful of labs have taken up this challenge and two basic strategies have emerged. The first involves redirecting endogenous ADAR to new sites by making editable structures using antisense RNA oligonucleotides. The second also utilizes antisense RNA oligonucleotides, but it uses them as guides to deliver the catalytic domain of engineered ADARs to new sites, much as CRISPR guides deliver Cas nucleases. In fact, despite the intense current focus on CRISPR-Cas9 genome editing, SDRE offers a number of distinct advantages. In the present review we will discuss these strategies in greater detail, focusing on the concepts on which they are based, how they were developed and tested, and their respective advantages and disadvantages. Though the precise and efficient re-direction of ADAR activity still remains a challenge, the systems that are being developed lay the foundation for SDRE as a powerful tool for transient genome editing.


Asunto(s)
Adenosina Desaminasa/genética , Sistemas CRISPR-Cas , Mutagénesis Sitio-Dirigida/métodos , Edición de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Adenosina/metabolismo , Adenosina Desaminasa/metabolismo , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Genoma Humano , Humanos , Inosina/metabolismo , Oligorribonucleótidos Antisentido/genética , Oligorribonucleótidos Antisentido/metabolismo , Dominios Proteicos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo
2.
RNA Biol ; 15(1): 104-114, 2018 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-29099293

RESUMEN

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.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutagénesis Sitio-Dirigida , Edición de ARN/genética , Transcriptoma/genética , Adenosina/genética , Codón sin Sentido/genética , Células Epiteliales/metabolismo , Humanos , Inosina/genética , Mutación/genética , ARN Guía de Kinetoplastida/genética , Transfección
3.
Nucleic Acids Res ; 44(21): e157, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27557710

RESUMEN

Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a U A: G premature termination codon (PTC) to tryptophan (U G: G) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.


Asunto(s)
Técnicas Genéticas , Edición de ARN , ARN Mensajero/genética , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Calibración , Codón sin Sentido , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Mutación , Péptidos/genética , Pichia/genética , Dominios Proteicos , ARN Guía de Kinetoplastida/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Triptófano/genética
4.
Proc Natl Acad Sci U S A ; 110(45): 18285-90, 2013 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-24108353

RESUMEN

Adenosine deaminases that act on RNA are a conserved family of enzymes that catalyze a natural process of site-directed mutagenesis. Biochemically, they convert adenosine to inosine, a nucleotide that is read as guanosine during translation; thus when editing occurs in mRNAs, codons can be recoded and the changes can alter protein function. By removing the endogenous targeting domains from human adenosine deaminase that acts on RNA 2 and replacing them with an antisense RNA oligonucleotide, we have engineered a recombinant enzyme that can be directed to edit anywhere along the RNA registry. Here we demonstrate that this enzyme can efficiently and selectively edit a single adenosine. As proof of principle in vitro, we correct a premature termination codon in mRNAs encoding the cystic fibrosis transmembrane conductance regulator anion channel. In Xenopus oocytes, we show that a genetically encoded version of our editase can correct cystic fibrosis transmembrane conductance regulator mRNA, restore full-length protein, and reestablish functional chloride currents across the plasma membrane. Finally, in a human cell line, we show that a genetically encoded version of our editase and guide RNA can correct a nonfunctional version of enhanced green fluorescent protein, which contains a premature termination codon. This technology should spearhead powerful approaches to correcting a wide variety of genetic mutations and fine-tuning protein function through targeted nucleotide deamination.


Asunto(s)
Adenosina Desaminasa/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Ingeniería Genética/métodos , Mutagénesis Sitio-Dirigida/métodos , Mutación/genética , Edición de ARN/genética , Adenosina Desaminasa/genética , Animales , Secuencia de Bases , Western Blotting , Codón sin Sentido/genética , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Datos de Secuencia Molecular , Oligonucleótidos/genética , Edición de ARN/fisiología , Xenopus
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