Improving Site‐Directed RNA Editing In Vitro and in Cell Culture by Chemical Modification of the GuideRNA

Vogel, P.; Schneider, Marius F.; Wettengel, Jacqueline; Stafforst, Thorsten

doi:10.1002/anie.201402634

Cited by 96 publications

(105 citation statements)

References 41 publications

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“…The hADAR1 deaminase domain and guide RNA were assembled by a rapid and specific single covalent bond between the SNAP-tag and BG moiety (Figure 1). After the guide RNA-deaminase conjugate was generated, it rapidly carried out site-directed specific RNA editing of the target mRNA, and the results showed the subsequent repair of a premature stop codon (UAG) into a tryptophan codon (UIG) in a fluorescent reporter gene, both in vitro and in human cells (293T) (69). Recently, the BG moiety was chemically masked using a light-sensitive 6-nitropiperonyloxymethyl (Npom) protection group, resulting in Npom-protected O6-benzylguanine (Npom-BG).…”

Section: Enzymatic Site-directed A-to-i Rna Editingmentioning

confidence: 99%

C-to-U editing and site-directed RNA editing for the correction of genetic mutations

Tsukahara

2017

BST

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Section: Enzymatic Site-directed A-to-i Rna Editingmentioning

confidence: 99%

C-to-U editing and site-directed RNA editing for the correction of genetic mutations

Tsukahara

2017

BST

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“…In one study, a guideRNA-dependent deaminase was constructed by engineering protein-guided human ADAR1 (hADAR1) into a guideRNA-dependent enzyme; the C-terminal catalytic domain of hADAR1 was fused to a SNAP-tag of the guideR-NA-dependent enzyme. The recombinant enzyme effectively and selectively repaired the stop66 nonsense mutation (UAG) to a functional tryptophan (UGG) (20). In another study, a recombinant site-directed editase was generated by removing the endogenous targeting domains of protein-guided hADAR2 and replacing them with an antisense RNA oligonucleotide; these enzymes were successfully used for directed, specific editing of the W496X mutation in cystic fibrosis transmembrane conductance regulator.…”

mentioning

confidence: 99%

Changing Blue Fluorescent Protein to Green Fluorescent Protein Using Chemical RNA Editing as a Novel Strategy in Genetic Restoration

Nguyen

Alam

et al. 2015

Chem Biol Drug Des

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Using the transition from cytosine of BFP (blue fluorescent protein) gene to uridine of GFP (green fluorescent protein) gene at position 199 as a model, we successfully controlled photochemical RNA editing to effect site-directed deamination of cytidine (C) to uridine (U). Oligodeoxynucleotides (ODNs) containing 5'-carboxyvinyl-2'-deoxyuridine ((CV) U) were used for reversible photoligation, and single-stranded 100-nt BFP DNA and in vitro-transcribed full-length BFP mRNA were the targets. Photo-cross-linking with the responsive ODNs was performed using UV (366 nm) irradiation, which was followed by heat treatment, and the cross-linked nucleotide was cleaved through photosplitting (UV, 312 nm). The products were analyzed using restriction fragment length polymorphism (RFLP) and fluorescence measurements. Western blotting and fluorescence-analysis results revealed that in vitro-translated proteins were synthesized from mRNAs after site-directed RNA editing. We detected substantial amounts of the target-base-substituted fragment using RFLP and observed highly reproducible spectra of the transition-GFP signal using fluorescence spectroscopy, which indicated protein stability. ODNc restored approximately 10% of the C-to-U transition. Thus, we successfully used non-enzymatic site-directed deamination for genetic restoration in vitro. In the near future, in vivo studies that include cultured cells and model animals will be conducted to treat genetic disorders.

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“…In other studies, the deaminase domain of human ADAR has been linked to a guide-RNA via either a SNAP-tag strategy or λN-boxB interaction (39,44–46). These methods depend on Watson and Crick base paring for target recognition and binding, in addition to the requirements for the delivery of chemically modified guide-RNA (for SNAP-tag) to living cells (46–48). Our dCas13a-hADAR2d system provides an attractive alternative for RNA editing applications.…”

Section: Discussionmentioning

confidence: 99%

“…P5-125 (PspCas13b), to mediate RNA editing in human cell lines has also been reported (38). With an improved hyperactive hADAR2d variant (E488Q), the efficiency of editing using the PspCas13b/hADAR2d(E488Q) system is comparable to or better than that of the guide-RNA approaches discussed above (43,46–48). The performance of our dCas13a-hADAR2d system in S. pombe varied for different targets, with an RNA editing efficiency of up to 54.5% being achieved for tdh1 (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing

Jing

Xie

Chen

et al. 2018

Nucleic Acids Research

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In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.

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Improving Site‐Directed RNA Editing In Vitro and in Cell Culture by Chemical Modification of the GuideRNA

Cited by 96 publications

References 41 publications

C-to-U editing and site-directed RNA editing for the correction of genetic mutations

C-to-U editing and site-directed RNA editing for the correction of genetic mutations

Changing Blue Fluorescent Protein to Green Fluorescent Protein Using Chemical RNA Editing as a Novel Strategy in Genetic Restoration

Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing

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