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

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

10.1101/2020.12.12.422520 ◽

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.

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Neurons expressing pathological Tau protein trigger dramatic changes in microglial morphology and dynamics

10.1101/731901 ◽

2019 ◽

Author(s):

Rahma Hassan-Abdi ◽

Alexandre Brenet ◽

Mohamed Bennis ◽

Constantin Yanicostas ◽

Nadia Soussi-Yanicostas

Keyword(s):

Morphological Changes ◽

Pathological Process ◽

Wild Type ◽

Zebrafish Model ◽

Genetic Ablation ◽

Microglial Morphology ◽

First Time ◽

The Brain ◽

Heterogeneous Class

AbstractMicroglial cells, the resident macrophages of the brain, are important players in the pathological process of numerous neurodegenerative disorders, including tauopathies, a heterogeneous class of diseases characterized by intraneuronal Tau aggregates. However, microglia response in Tau pathologies remains poorly understood. Here we exploit a genetic zebrafish model of tauopathy, combined with live microglia imaging, to investigate the behaviour of microglia in vivo in the disease context. Results show that while microglia were almost immobile and displayed long and highly dynamic branches in a wild-type context, in presence of diseased neurons cells became highly mobile and displayed morphological changes, with highly mobile cell bodies together with fewer and shorter processes. We also imaged, for the first time to our knowledge, the phagocytosis of apoptotic tauopathic neurons by microglia in vivo and observed that microglia engulfed about as twice materials as in controls. Finally, genetic ablation of microglia in zebrafish tauopathy model significantly increased Tau hyperphosphorylation, suggesting that microglia provide neuroprotection to diseased neurons. Our findings demonstrate for the first time the dynamics of microglia in contact with tauopathic neurons in vivo and open perspectives for the real-time study of microglia in many neuronal diseases.

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

Science Translational Medicine ◽

10.1126/scitranslmed.aay9101 ◽

2020 ◽

Vol 12 (546) ◽

pp. eaay9101 ◽

Cited By ~ 11

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.

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

Nature Biotechnology ◽

10.1038/s41587-021-00933-4 ◽

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.

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

eLife ◽

10.7554/elife.65552 ◽

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.

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Pyclen Based Ln(III) Complexes as Highly Luminescent Bioprobes for in Vitro and in Vivo 1P and 2P Bioimaging Applications

10.26434/chemrxiv.12047532 ◽

2020 ◽

Author(s):

Nadège Hamon ◽

Amandine Roux ◽

Maryline Beyler ◽

Jean-Christophe Mulatier ◽

Chantal Andraud ◽

...

Keyword(s):

High Performance ◽

Breast Cancer Cells ◽

Positive Impact ◽

Quantum Yields ◽

Human Breast ◽

Zebrafish Model ◽

High Brightness ◽

First Time

Two pyclen based lanthanide chelators, L4b and L4c, bearing two specific picolinate 2P antennas (tailor-made for each targeted metal) and one acetate arm arranged in a dissymmetrical manner, have been synthesized to form, with the already described ligand L4a, a complete family of lanthanide luminescent bioprobes: [EuL4a], [SmL4a], [YbL4b], [TbL4c] and [DyL4c]. Additionally, symmetrically arranged regioisomer L4a’ was also synthesized as well as its [EuL4a’] complex to highlight the astonishing positive impact of the dissymmetrical N-distribution of the functional chelating arm. The investigation clearly shows the high performance of each bioprobe, which, depending on the complexed lanthanide, could be used in various applications. Each presents high brightness, quantum yields and lifetimes. Staining of the complexes into living human breast cancer cells was observed. In addition, in vivo 2P-microscopy was performed for the first time on a living Zebrafish model with [EuL4a]. No apparent toxicity was detected on the growth of the zebrafish and images of high quality were obtained.

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Alexander Disease Modeling in Zebrafish: An In Vivo System Suitable to Perform Drug Screening

Genes ◽

10.3390/genes11121490 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1490

Author(s):

Simona Candiani ◽

Silvia Carestiato ◽

Andreas F. Mack ◽

Daniele Bani ◽

Matteo Bozzo ◽

...

Keyword(s):

Heat Shock ◽

Drug Screening ◽

Disease Modeling ◽

De Novo ◽

Alexander Disease ◽

Glial Fibrillary Acid Protein ◽

Suitable Model ◽

Zebrafish Model ◽

In Vivo Models

Alexander disease (AxD) is a rare astrogliopathy caused by heterozygous mutations, either inherited or arising de novo, on the glial fibrillary acid protein (GFAP) gene (17q21). Mutations in the GFAP gene make the protein prone to forming aggregates which, together with heat-shock protein 27 (HSP27), αB-crystallin, ubiquitin, and proteasome, contribute to form Rosenthal fibers causing a toxic effect on the cell. Unfortunately, no pharmacological treatment is available yet, except for symptom reduction therapies, and patients undergo a progressive worsening of the disease. The aim of this study was the production of a zebrafish model for AxD, to have a system suitable for drug screening more complex than cell cultures. To this aim, embryos expressing the human GFAP gene carrying the most severe p.R239C under the control of the zebrafish gfap gene promoter underwent functional validation to assess several features already observed in in vitro and other in vivo models of AxD, such as the localization of mutant GFAP inclusions, the ultrastructural analysis of cells expressing mutant GFAP, the effects of treatments with ceftriaxone, and the heat shock response. Our results confirm that zebrafish is a suitable model both to study the molecular pathogenesis of GFAP mutations and to perform pharmacological screenings, likely useful for the search of therapies for AxD.

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Recent Advances in CRISPR/Cas9-Based Genome Editing Tools for Cardiac Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms222010985 ◽

2021 ◽

Vol 22 (20) ◽

pp. 10985

Author(s):

Juliët Schreurs ◽

Claudia Sacchetto ◽

Robin M. W. Colpaert ◽

Libero Vitiello ◽

Alessandra Rampazzo ◽

...

Keyword(s):

Genome Editing ◽

Disease Modeling ◽

Zinc Finger Nucleases ◽

Cardiac Diseases ◽

Transcription Activator ◽

Base Editing ◽

Epochal Change ◽

Recent Developments ◽

Dna Editing ◽

First Time

In the past two decades, genome editing has proven its value as a powerful tool for modeling or even treating numerous diseases. After the development of protein-guided systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which for the first time made DNA editing an actual possibility, the advent of RNA-guided techniques has brought about an epochal change. Based on a bacterial anti-phage system, the CRISPR/Cas9 approach has provided a flexible and adaptable DNA-editing system that has been able to overcome several limitations associated with earlier methods, rapidly becoming the most common tool for both disease modeling and therapeutic studies. More recently, two novel CRISPR/Cas9-derived tools, namely base editing and prime editing, have further widened the range and accuracy of achievable genomic modifications. This review aims to provide an overview of the most recent developments in the genome-editing field and their applications in biomedical research, with a particular focus on models for the study and treatment of cardiac diseases.

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Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2

Science Advances ◽

10.1126/sciadv.aaz8534 ◽

2020 ◽

Vol 6 (27) ◽

pp. eaaz8534

Author(s):

Anindya Dey ◽

Shubhangi Prabhudesai ◽

Yushan Zhang ◽

Geeta Rao ◽

Karthikeyan Thirugnanam ◽

...

Keyword(s):

Zinc Finger ◽

Hypoxia Inducible Factor ◽

Point Mutations ◽

Prolyl Hydroxylase ◽

Zinc Finger Motif ◽

Hydroxylase Activity ◽

Zebrafish Model ◽

Enzymatic Action

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine β-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

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Efficient precise in vivo base editing in adult dystrophic mice

Nature Communications ◽

10.1038/s41467-021-23996-y ◽

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.

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