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 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 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 In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness

2020 ◽  
Vol 12 (546) ◽  
pp. eaay9101 ◽  
Author(s):  
Wei-Hsi Yeh ◽  
Olga Shubina-Oleinik ◽  
Jonathan M. Levy ◽  
Bifeng Pan ◽  
Gregory A. Newby ◽  
...  
Keyword(s):  
Hair Cell ◽  
Point Mutation ◽  
Hair Cells ◽  
Point Mutations ◽  
Sensory Transduction ◽  
Auditory Function ◽  
Sensory Hair ◽  
Base Editing ◽  
Sensory Hair Cells

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild-type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.

scholarly journals In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

2021 ◽  
Author(s):  
Tanja Rothgangl ◽  
Melissa K. Dennis ◽  
Paulo J. C. Lin ◽  
Rurika Oka ◽  
Dominik Witzigmann ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Point Mutations ◽  
Density Lipoprotein ◽  
Negative Regulator ◽  
Base Editing ◽  
Guide Rna ◽  
Humoral Immune ◽  
Cholesterol Levels ◽  
Adenine Base

AbstractMost known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

Recent Advances in CRISPR/Cas9 Directed Base Editing

2021 ◽  
Vol 21 ◽  
Author(s):  
Nan Liu ◽  
Lifang Zhou ◽  
Junyan , Qu ◽  
Shaohua Yao
Keyword(s):  
Recent Progress ◽  
High Efficiency ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Clinical Applications ◽  
Great Promise ◽  
Base Editing ◽  
Recent Advances ◽  
Dna Base ◽  
Genomic Editing

: Recently, CRISPR based techniques had significantly improved our ability to make desired changes and regulations in various genomes. Among them, targeted base editing is one of the most powerful techniques in making precise genomic editing. Base editing enabled irreversible conversion of specific single DNA base, from C to T or and from A to G, in desired genomic loci. This technique has important implications in the study of human genetic diseases, considering that many of them resulted from point mutations. More importantly, high efficiency of those editing tools also provided great promise in clinical applications. In this review, we discuss recent progress and challenges of base editing tools.

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 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.

Abstract 55: Disruption of PCSK9 Using a Targeted Base Editing Strategy

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Alexandra C Chadwick ◽  
Kiran Musunuru
Keyword(s):  
Genome Editing ◽  
Rna Editing ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Lower Efficiency ◽  
Nonsense Mutations ◽  
Base Editing ◽  
Stop Codons ◽  
Editing Domain

Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases blood low-density lipoprotein (LDL) cholesterol by acting as an LDL receptor antagonist, thereby impairing LDL particle clearance. Since genetic disruption of PCSK9 is linked to reduced risk of coronary heart disease (CHD), our recent work has sought to permanently knock out the gene by using new genome editing technology. Recently reported “base editors” introduce point mutations at specific locations in the genome without the need for DNA double-strand breaks and, thus, with a lowered incidence of off-target effects. These base editors build on the CRISPR-Cas9 system by tethering an RNA-editing domain to a nickase version of Cas9, allowing for specific CT and/or GA base alterations. In this study, we used the “BE3” base editor (which uses the APOBEC-1 RNA-editing domain) to specifically target codons encoding tryptophans (TGG) or glutamines (CAG or CAA) to introduce nonsense mutations (producing stop codons TAG, TGA, or TAA) into human PCSK9 . The number of testable targets was increased by introducing specific point mutations into the BE3 construct (D1135V, R1335Q, T1337R in the Cas9 portion of BE3) to alter the protospacer adjacent motif (PAM) from NGG to NGA. Using HEK293 cells, we individually targeted a number of codons spanning the first seven exons in PCSK9 and identified several efficient targets. Most notably, the codons encoding glutamine 278 and glutamine 302 could be altered to stop codons in ~50% of alleles, as determined by the CEL-I nuclease mismatch assay and Sanger sequencing. We then targeted glutamine 278 in human induced pluripotent stem cells (iPSCs) and demonstrated base editing to introduce nonsense mutations into PCSK9 , albeit at a lower efficiency than in HEK293 cells. As a next step towards translation to human patients, we are targeting Pcsk9 in the mouse liver in vivo with BE3. Base editing may prove to be an efficient, safer strategy than standard CRISPR-Cas9 genome editing and holds promise as a strategy for the prevention of CHD.

scholarly journals Disease modeling by efficient genome editing using a near PAM-less base editor in vivo.

2021 ◽  
Author(s):  
Marion Rosello ◽  
Malo Serafini ◽  
Marina Mione ◽  
Jean-Paul Concordet ◽  
Filippo Del Bene
Keyword(s):  
Disease Modeling ◽  
Point Mutations ◽  
Selection Method ◽  
Innovative Technology ◽  
Zebrafish Model ◽  
Base Editing ◽  
Protospacer Adjacent Motif ◽  
Visual Screening ◽  
First Time

Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing the NRN PAM sequence, we could introduce point mutations for the first time in an animal model and achieved up to 100% efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we applied our method to create a new zebrafish model for melanoma predisposition based on the simultaneous editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.

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