A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1

Wang, Yuru; Havel, Jocelyn; Beal, Peter A.

doi:10.1021/acschembio.5b00711

“…Activity by the HyperADARcd alone is a likely candidate, and we are aware of editing by the human HyperADAR1-cd in other systems (Wang et al 2015). However, we never obtained any substantial editing with expression of only the Drosophila HyperADARcd (e.g., Fig.…”

Section: Discussionmentioning

confidence: 88%

Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein

Xu

¹

,

Rahman

²

,

Rosbash

³

2017

RNA

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We previously developed TRIBE, a method for the identification of cell-specific RNA-binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA-editing enzyme ADAR and performs adenosine-to-inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.

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“…Might there be a comparable source of HyperTRIBE false positives? Activity by the HyperADARcd alone is a likely candidate, and we are aware of editing by the human HyperADAR1-cd in other systems (Wang et al 2015). However, we never obtained any substantial editing with expression of only the Drosophila HyperADARcd (e.g., Fig.…”

Section: Discussionmentioning

confidence: 88%

Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein

Xu

¹

,

Rahman

²

,

Rosbash

³

2017

Preprint

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We previously developed TRIBE, a method for the identification of cell-specific RNA-binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA-editing enzyme ADAR and performs adenosine-to-inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.

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“…2A). To enhance target adenosine deamination rates we introduced two additional modifications to our initial RNA editing design: we introduced a mismatched cytidine opposite the target adenosine, which has been previously reported to increase deamination frequency, and fused dCas13b with the deaminase domains of human ADAR1 or ADAR2 containing hyperactivating mutations to enhance catalytic activity (ADAR1 DD (E1008Q) (27) or ADAR2 DD (E488Q) (21)).…”

Section: Cas13-adar Fusions Enable Targeted Rna Editingmentioning

confidence: 99%

RNA editing with CRISPR-Cas13

Cox

¹

,

Gootenberg

²

,

Abudayyeh

³

et al. 2017

Science

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Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided RNases Cas13. Here, we profile Type VI systems to engineer a Cas13 ortholog capable of robust knockdown and demonstrate RNA editing by using catalytically-inactive Cas13 (dCas13) to direct adenosine to inosine deaminase activity by ADAR2 to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineer this system to create a high specificity variant, REPAIRv2, that is 919 times more specific than REPAIRv1 as well as minimize the system to ease viral delivery. REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology.

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A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1

Cited by 26 publications

References 33 publications

Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein

Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein

Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein

RNA editing with CRISPR-Cas13

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