RNA-Guided Adenosine Deaminases: Advances and Challenges for Therapeutic RNA Editing

Chen, Genghao; Katrekar, Dhruva; Mali, Prashant

doi:10.1021/acs.biochem.9b00046

Cited by 20 publications

(17 citation statements)

References 107 publications

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“…Different SDRE promising platforms have been reported in recent literature [14][15][16][17]. Some are based on recruitment of native adenosine deaminases acting on RNA (ADARs) by antisense oligonucleotides (ASOs) [18][19][20], others on the recruitment of exogenous recombinant ADARs via the fusion to an RNA guide recognizing domain.…”

Section: Introductionmentioning

confidence: 99%

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

Melfi

Chiavetta

Barra

et al. 2020

IJMS

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Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool “REPAIRv2” (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in different cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFPopal and CFTRW1282X, respectively.

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Section: Introductionmentioning

confidence: 99%

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

Melfi

Chiavetta

Barra

et al. 2020

IJMS

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“…Regarding the location of the target whether it should be in the 5′ (as we have done) or middle or 3′ position of the guideRNA, we have tried with different positions and we have found that the best efficiency we have got from the 5′ position (7th position in 21 bp guideRNA) of the target in the guideRNA (Supplementary datda 5 ). Katrekar et al (2019) also applied the guideRNA of 20 bp length and they placed the mismatch (at the site of target) at 6th position in 20 bp guideRNA 20 , 46 .…”

Section: Methodsmentioning

confidence: 99%

RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code

Bhakta

Sakari

Tsukahara

2020

Sci Rep

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Many genetic diseases are caused by T-to-C point mutations. Hence, editing of mutated genes represents a promising strategy for treating these disorders. We engineered an artificial RNA editase by combining the deaminase domain of APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1) with a guideRNA (gRNA) which is complementary to target mRNA. In this artificial enzyme system, gRNA is bound to MS2 stem-loop, and deaminase domain, which has the ability to convert mutated target nucleotide C-to-U, is fused to MS2 coat protein. As a target RNA, we used RNA encoding blue fluorescent protein (BFP) which was derived from the gene encoding GFP by 199 T > C mutation. Upon transient expression of both components (deaminase and gRNA), we observed GFP by confocal microscopy, indicating that mutated 199C in BFP had been converted to U, restoring original sequence of GFP. This result was confirmed by PCR–RFLP and Sanger’s sequencing using cDNA from transfected cells, revealing an editing efficiency of approximately 21%. Although deep RNA sequencing result showed some off-target editing events in this system, we successfully developed an artificial RNA editing system using artificial deaminase (APOBEC1) in combination with MS2 system could lead to therapies that treat genetic disease by restoring wild-type sequence at the mRNA level.

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“…Several systems were recently developed to recruit ADAR enzymes to specific sites for site-directed RNA editing [23][24][25][26][27][28][29] , providing novel tools to study biological function and a safer and reversible alternative to gene therapy 23,[47][48][49] . These RNA engineering approaches use antisense RNA oligoribonucleotides as guide RNAs (gRNAs) to recruit either engineered or endogenous ADAR enzymes.…”

Section: Discussionmentioning

confidence: 99%

Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis

Liu

Sun

Shcherbina

et al. 2019

Preprint

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Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes occurs in double-stranded RNAs (dsRNAs). How the RNA sequence and structure (i.e., the cis-regulation) determine the editing efficiency and specificity is poorly understood, despite a compelling need towards functional understanding of known editing events and transcriptome engineering of desired adenosines. We developed a CRISPR/Cas9-mediated saturation mutagenesis approach to generate comprehensive libraries of point mutations near an editing site and its editing complementary sequence (ECS) at the endogenous genomic locus. We used machine learning to integrate diverse RNA sequence features and computationally predicted structures to model editing levels measured by deep sequencing and identified cis-regulatory features of RNA editing. As proof-of-concept, we applied this integrative approach to three editing substrates. Our models explained over 70% of variation in editing levels. The models indicate that RNA sequence and structure features synergistically determine the editing levels.Our integrative approach can be broadly applied to any editing site towards the goal of deciphering the RNA editing code. It also provides guidance for designing and screening of antisense RNA sequences that form dsRNA duplex with the target transcript for ADAR-mediated transcriptome engineering.

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RNA-Guided Adenosine Deaminases: Advances and Challenges for Therapeutic RNA Editing

Cited by 20 publications

References 107 publications

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code

Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis

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