An RNA–Deaminase Conjugate Selectively Repairs Point Mutations

Stafforst, Thorsten; Schneider, Marius F.

doi:10.1002/anie.201206489

Cited by 136 publications

(109 citation statements)

References 22 publications

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“…Full A-to-I conversion at the targeted site (A200) was achieved (Figure 1B, (a)). However, in contrast to our engineered SNAP-ADAR deaminase, (34,42) wild-type ADAR2 was dramatically more reactive and gave massive off-target editing, with full conversion at A381 (Figure 1B, (a)), 50–70% yield at A295, A380, A476 and minor editing at various sites (Supplementary Figure S2). This off-site editing was typically guideRNA-independent.…”

Section: Resultsmentioning

confidence: 66%

“…A reporter mRNA (cyan fluorescent protein) that contains a single G-to-A point mutation generating a nonsense stop signal (Trp 66 →amber) served as substrate (Figure 1), as described before (34,42). Wild-type human ADAR2 was expressed and purified from yeast (YVH10), as described before (42).…”

Section: Resultsmentioning

confidence: 99%

“…However, the main issue of low efficiency and in particular off-site editing in the guideRNA/mRNA could not be solved at that time. In 2012 and 2013, we (34,35) and the lab of Joshua Rosenthal (36) have reanimated the concept of site-directed RNA editing by independent engineering of artificial editing enzymes that address the catalytic activity in a highly rational way with the help of external guideRNAs. Such strategies work inside mammalian cell culture and even in a simple organism (37) and allow the repair of disease-relevant genes, like the CFTR (36) mRNA.…”

Section: Introductionmentioning

confidence: 99%

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Harnessing human ADAR2 for RNA repair – Recoding a PINK1 mutation rescues mitophagy

et al. 2016

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Site-directed A-to-I RNA editing is a technology for re-programming genetic information at the RNA-level. We describe here the first design of genetically encodable guideRNAs that enable the re-addressing of human ADAR2 toward specific sites in user-defined mRNA targets. Up to 65% editing yield has been achieved in cell culture for the recoding of a premature Stop codon (UAG) into tryptophan (UIG). In the targeted gene, editing was very specific. We applied the technology to recode a recessive loss-of-function mutation in PINK1 (W437X) in HeLa cells and showed functional rescue of PINK1/Parkin-mediated mitophagy, which is linked to the etiology of Parkinson's disease. In contrast to other editing strategies, this approach requires no artificial protein. Our novel guideRNAs may allow for the development of a platform technology that requires only the administration or expression of a guideRNA to recode genetic information, with high potential for application in biology and medicine.

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Section: Resultsmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Harnessing human ADAR2 for RNA repair – Recoding a PINK1 mutation rescues mitophagy

et al. 2016

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“…By introducing RNA oligonucleotides complementary to a premature termination codon, Woolf et al induced endogenous ADAR to nonspecifically edit the region, including the premature termination codon, both in vitro and in Xenopus embryos (33). A recent study has reported a more directed approach, similar to our own (34). In it the authors coupled the catalytic domain of human ADAR1 to a guide RNA oligonucleotide using an in vitro reaction.…”

Section: Discussionmentioning

confidence: 99%

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

Montiel-González

Vallecillo-Viejo

Yudowski

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

198

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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 fulllength 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.

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“…Guide-RNAs not only allow mRNA target specificity but more importantly induce the formation of secondary structures necessary for achieving highly efficient and selective targeting of a single adenosine, without the risk of over-editing. Additional studies have shown efficient in vitro correction of a stop codon introduced into a fluorescent protein, and at CFTR W496X and EGFP W58X, by using a similar approach coupling the catalytic domain of ADAR1 to a guideRNA [88,89]. Although these studies improved the prospect of directed RNA editing for in vivo applications and medicine, many parameters remain without optimization, including mode of delivery.…”

Section: Concluding Remarks and Future Directionsmentioning

confidence: 99%

RNA rewriting, recoding, and rewiring in human disease

Zipeto

Jiang

Melese

et al. 2015

Trends in Molecular Medicine

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An RNA–Deaminase Conjugate Selectively Repairs Point Mutations

Abstract: Checking for mistakes: By conjugating a catalytic domain with a guide RNA, deamination activity can be harnessed to repair a specific codon on mRNA. This method can be used for the highly selective repair of point mutations in mRNA by site-selective editing.

Cited by 136 publications

References 22 publications

Harnessing human ADAR2 for RNA repair – Recoding a PINK1 mutation rescues mitophagy

Harnessing human ADAR2 for RNA repair – Recoding a PINK1 mutation rescues mitophagy

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

RNA rewriting, recoding, and rewiring in human disease

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