scholarly journals Efficient precise in vivo base editing in adult dystrophic mice

2020 ◽  
Author(s):  
Li Xu ◽  
Chen Zhang ◽  
Haiwen Li ◽  
Peipei Wang ◽  
Yandi Gao ◽  
...  
Keyword(s):  
Rna Editing ◽  
Deep Sequencing ◽  
High Efficiency ◽  
Contractile Function ◽  
Functional Improvement ◽  
Base Editing ◽  
Monogenic Diseases ◽  
Editing Activity ◽  
Target Rna

ABSTRACTBackgroundRecent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.MethodsWe employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.ResultsWe showed efficient in vitro base correction of the dystrophin mutation carried in mdx4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected.ConclusionsThis study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.

scholarly journals Efficient precise in vivo base editing in adult dystrophic mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Li Xu ◽  
Chen Zhang ◽  
Haiwen Li ◽  
Peipei Wang ◽  
Yandi Gao ◽  
...  
Keyword(s):  
High Efficiency ◽  
Functional Improvement ◽  
Systemic Delivery ◽  
Mutation Site ◽  
Base Editing ◽  
Adult Mice ◽  
Exciting Opportunity ◽  
Protospacer Adjacent Motif ◽  
Monogenic Diseases

AbstractRecent advances in base editing have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) nearby the mutation site further limits the targeting feasibility. Here we modify the NG-targeting adenine base editor (iABE-NGA) to overcome these challenges and demonstrate the high efficiency to precisely edit a Duchenne muscular dystrophy (DMD) mutation in adult mice. Systemic delivery of AAV9-iABE-NGA results in dystrophin restoration and functional improvement. At 10 months after AAV9-iABE-NGA treatment, a near complete rescue of dystrophin is measured in mdx4cv mouse hearts with up to 15% rescue in skeletal muscle fibers. The off-target activities remains low and no obvious toxicity is detected. This study highlights the promise of permanent base editing using iABE-NGA for the treatment of monogenic diseases.

scholarly journals Precise adenine base editors that exhibit minimized cytosine catalysis

2020 ◽  
Author(s):  
You Kyeong Jeong ◽  
SeokHoon Lee ◽  
Gue-Ho Hwang ◽  
Sung-Ah Hong ◽  
Se-eun Park ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Adenosine Deaminase ◽  
Rna Editing ◽  
The Other ◽  
Cytosine Deamination ◽  
Base Editing ◽  
Target Dna ◽  
Editing Activity ◽  
Target Rna ◽  
Adenine Base

Abstract Adenine base editors (ABEs) promise specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target DNA site and exhibit transcriptome-wide off-target RNA editing. To alleviate the ABE-mediated cytosine editing activity, here we engineered the commonly-used version of adenosine deaminase, TadA7.10, to contain rationally designed mutations. We ultimately found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited greatly reduced cytosine deamination activity, and conversely, ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity rather than adenine editing. We found that the D108Q mutation also reduces cytosine deamination activity in two recently-developed versions of ABE, ABE8e and ABE8s, and has a synergistic effect with V106W, a key mutation that reduces off-target RNA editing. On the other hand, by incorporating the P48R mutation into ABE7.10, we demonstrated TC-specific base editing tools that enable either TC-to-TT or TC-to-TG conversions, broadening the utility of base editors.

scholarly journals Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

2002 ◽  
Vol 22 (19) ◽  
pp. 6726-6734 ◽  
Author(s):  
Tetsuya Miyamoto ◽  
Junichi Obokata ◽  
Masahiro Sugiura
Keyword(s):  
Rna Editing ◽  
Editing Site ◽  
Mrna Editing ◽  
Higher Plant ◽  
In Vitro System ◽  
Cis Acting ◽  
Biochemical Identification ◽  
Editing Activity

ABSTRACT RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

scholarly journals SUMO-1 Modification Alters ADAR1 Editing Activity

2005 ◽  
Vol 16 (11) ◽  
pp. 5115-5126 ◽  
Author(s):  
Joana M.P. Desterro ◽  
Liam P. Keegan ◽  
Ellis Jaffray ◽  
Ron T. Hay ◽  
Mary A. O'Connell ◽  
...  
Keyword(s):  
Rna Editing ◽  
Nucleolar Localization ◽  
Dense Fibrillar Component ◽  
Wild Type ◽  
Fibrillar Center ◽  
Editing Activity ◽  
Fibrillar Component ◽  
Editing Enzyme

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.

scholarly journals Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

2020 ◽  
Author(s):  
Marion Rosello ◽  
Juliette Vougny ◽  
François Czarny ◽  
Marina Mione ◽  
Jean-Paul Concordet ◽  
...  
Keyword(s):  
High Efficiency ◽  
Human Gene ◽  
Point Mutations ◽  
Base Change ◽  
Model System ◽  
Human Genetic Disease ◽  
Base Editing ◽  
Oncogenic Mutations ◽  
Cell Signaling Pathways

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes among which cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

scholarly journals Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Marion Rosello ◽  
Juliette Vougny ◽  
François Czarny ◽  
Marina C Mione ◽  
Jean-Paul Concordet ◽  
...  
Keyword(s):  
High Efficiency ◽  
Human Gene ◽  
Point Mutations ◽  
Base Change ◽  
Model System ◽  
Human Genetic Disease ◽  
Base Editing ◽  
Oncogenic Mutations ◽  
Cell Signaling Pathways

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

scholarly journals Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

2020 ◽  
Author(s):  
Dhruva Katrekar ◽  
Nathan Palmer ◽  
Yichen Xiang ◽  
Anushka Saha ◽  
Dario Meluzzi ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Motifs ◽  
Mutagenesis Screen ◽  
Base Editing ◽  
Adenosine Deaminases ◽  
Editing Activity ◽  
Exogenous Delivery ◽  
Deaminase Domain ◽  
The Impact ◽  
Increased Activity

ABSTRACTAdenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5’ guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5’-GA-3’ motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

scholarly journals Development of a Single Construct System for Site-Directed RNA Editing Using MS2-ADAR

2020 ◽  
Vol 21 (14) ◽  
pp. 4943
Author(s):  
Tetsuto Tohama ◽  
Matomo Sakari ◽  
Toshifumi Tsukahara
Keyword(s):  
Gene Therapy ◽  
Rna Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Cultured Cells ◽  
Point Mutations ◽  
Gene Repair ◽  
Guide Rnas ◽  
Single Construct

Site-directed RNA editing (SDRE) technologies have great potential for treating genetic diseases caused by point mutations. Our group and other researchers have developed SDRE methods utilizing adenosine deaminases acting on RNA (ADARs) and guide RNAs recruiting ADARs to target RNAs bearing point mutations. In general, efficient SDRE relies on introducing numerous guide RNAs relative to target genes. However, achieving a large ratio is not possible for gene therapy applications. In order to achieve a realistic ratio, we herein developed a system that can introduce an equal number of genes and guide RNAs into cultured cells using a fusion protein comprising an ADAR fragment and a plasmid vector containing one copy of each gene on a single construct. We transfected the single construct into HEK293T cells and achieved relatively high efficiency (up to 42%). The results demonstrate that efficient SDRE is possible when the copy number is similar for all three factors (target gene, guide RNA, and ADAR enzyme). This method is expected to be capable of highly efficient gene repair in vivo, making it applicable for gene therapy.

scholarly journals DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis

2021 ◽  
Vol 4 (6) ◽  
pp. 510-522
Author(s):  
Mizuki Takenaka ◽  
Sachi Takenaka ◽  
Tatjana Barthel ◽  
Brody Frink ◽  
Sascha Haag ◽  
...  
Keyword(s):  
Rna Editing ◽  
Ground State ◽  
Cytidine Deaminase ◽  
Pentatricopeptide Repeat ◽  
Domain Structures ◽  
Differential Scanning Fluorimetry ◽  
Pentatricopeptide Repeat Proteins ◽  
Cytidine Deamination ◽  
Target Rna

AbstractRNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts—mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis.

scholarly journals In vivo adenine base editing corrects newborn murine model of Hurler syndrome

2021 ◽  
Author(s):  
Jing Su ◽  
Xiu Jing ◽  
Kai qin She ◽  
Xiao mei Zhong ◽  
Qin yu Zhao ◽  
...  
Keyword(s):  
Murine Model ◽  
Human Condition ◽  
Type I ◽  
Newborn Mice ◽  
Base Editing ◽  
Minus Sign ◽  
Monogenic Diseases ◽  
Mps I ◽  
Adenine Base

Mucopolysaccharidosis type I (MPS I) is a severe disease caused by loss-of-function mutations variants in the α-L-iduronidase (IDUA) gene. In vivo genome editing represents a promising strategy to correct IDUA mutations, and has the potential to permanently restore IDUA function over the lifespan of the patients. Here, we used adenine base editing to directly convert A>G (TAG>TGG) in newborn murine model harboring Idua-W392X mutation, which recapitulates the human condition and is analogous to the highly prevalent human W402X mutation. We engineered a split-intein dual-adeno-associated virus (AAV) 9 in vivo adenine base editor to circumvent the package size limit of AAV vectors. Intravenous injection of AAV9-base editor system into MPS I newborn mice led to sustained enzyme expression sufficient for correction of metabolic disease (GAGs substrate accumulation) and prevention of neurobehavioral deficits. We observed a reversion of the W392X mutation in 22.46 plus-or-minus sign 6.74% of hepatocytes, 11.18 plus-or-minus sign 5.25% of heart and 0.34 plus-or-minus sign 0.12% of brain, along with decreased GAGs storage in peripheral organs (liver, spleen, lung and kidney). Collectively, these data showed the promise of a base editing approach to precisely correct a common genetic cause of MPS I in vivo and could be broadly applicable to the treatment of a wide array of monogenic diseases.

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