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Archives de Pediatrie : Organe Officiel... Feb 2020Pathogenic variants of the CFTR gene are responsible for a broad phenotypic spectrum characterized by malfunction of some exocrine tissues, with an autosomal recessive... (Review)
Review
Pathogenic variants of the CFTR gene are responsible for a broad phenotypic spectrum characterized by malfunction of some exocrine tissues, with an autosomal recessive mode of inheritance. More than 2,000 variants, distributed throughout the CFTR gene, have been identified, with different effects on the gene and protein expression and function. Genotype-phenotype correlation studies have associated severe variants with a typical multi-organ form of cystic fibrosis, while mild variants are involved in monosymptomatic or adult-onset diseases, called CFTR-related disorders. However, the interpretation of rare variants remains challenging. This review presents an overview of the epidemiology of CFTR variants worldwide and in France and describes the functional classification. Finally, some frequent cystic fibrosis-causing and mild CFTR variants are used as example to depict the molecular pathology of the CFTR locus. Finally, we give the recommendations concerning nomenclature and classification that are useful for appropriate genetic counseling. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.
Topics: Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Exons; Gene Rearrangement; Humans; Inteins; Mutation; Phenotype; RNA, Untranslated
PubMed: 32172939
DOI: 10.1016/S0929-693X(20)30044-0 -
Computational and Structural... 2019Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have been employed as a powerful versatile technology for... (Review)
Review
Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have been employed as a powerful versatile technology for programmable gene editing, transcriptional modulation, epigenetic modulation, and genome labeling, Yet better control of their activity is important to accomplish greater precision and to reduce undesired outcomes such as off-target events. The use of small molecules to control CRISPR/Cas activity represents a promising direction. Here, we provide an updated review on multiple drug inducible CRISPR/Cas systems and discuss their distinct properties. We arbitrarily divided the emerging drug inducible CRISPR/Cas systems into two categories based on whether at transcription or protein level does chemical control occurs. The first category includes Tet-On/Off system and Cre-dependent system. The second category includes chemically induced proximity systems, intein splicing system, 4-Hydroxytamoxifen-Estrogen Receptor based nuclear localization systems, allosterically regulated Cas9 system, and destabilizing domain mediated protein degradation systems. Finally, the advantages and limitations of each system were summarized.
PubMed: 31462973
DOI: 10.1016/j.csbj.2019.07.015 -
Science Translational Medicine Mar 2022Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in...
Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Cas9 prime editor (PE) lacking the RNaseH domain (PE2) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the locus and were further elevated to 58% by delivering unsplit PE2 via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.
Topics: Animals; Dependovirus; Gene Editing; Liver Diseases; Mice; Phenylketonurias
PubMed: 35294257
DOI: 10.1126/scitranslmed.abl9238 -
Molecular Therapy : the Journal of the... Sep 2022Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase...
Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.
Topics: Animals; CRISPR-Cas Systems; Dependovirus; Gene Editing; Genetic Vectors; Mice; Proprotein Convertase 9; RNA, Guide, CRISPR-Cas Systems; RNA-Directed DNA Polymerase
PubMed: 35808824
DOI: 10.1016/j.ymthe.2022.07.001 -
Molecular Cell Apr 2022Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment....
Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.
Topics: Amino Acids, Essential; Animals; Diet, High-Fat; Hepatocytes; Liver; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Ubiquitination
PubMed: 35245436
DOI: 10.1016/j.molcel.2022.01.021 -
PLoS Pathogens Mar 2021Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent,...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.
Topics: Antiviral Agents; COVID-19; Cell Culture Techniques; Containment of Biohazards; Genome, Viral; High-Throughput Screening Assays; Humans; SARS-CoV-2; Virus Replication
PubMed: 33711082
DOI: 10.1371/journal.ppat.1009439 -
Nature Apr 2023Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in...
Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.
Topics: Humans; Cell Nucleus; Chromatin; Cross-Linking Reagents; Energy Transfer; Epigenomics; Inteins; Iridium; Mutation; Neoplasms; Photosensitizing Agents; Protein Binding; Protein Interaction Maps
PubMed: 37020029
DOI: 10.1038/s41586-023-05914-y -
Nature Biomedical Engineering Nov 2022The viral delivery of base editors has been complicated by their size and by the limited packaging capacity of adeno-associated viruses (AAVs). Typically, dual-AAV...
The viral delivery of base editors has been complicated by their size and by the limited packaging capacity of adeno-associated viruses (AAVs). Typically, dual-AAV approaches based on trans-splicing inteins have been used. Here we show that, compared with dual-AAV systems, AAVs with size-optimized genomes incorporating compact adenine base editors (ABEs) enable efficient editing in mice at similar or lower doses. Single-AAV-encoded ABEs retro-orbitally injected in mice led to editing efficiencies in liver (66%), heart (33%) and muscle (22%) tissues that were up to 2.5-fold those of dual-AAV ABE8e, and to a 93% knockdown (on average) of human PCSK9 and of mouse Pcsk9 and Angptl3 in circulation, concomitant with substantial reductions of plasma cholesterol and triglycerides. Moreover, three size-minimized ABE8e variants, each compatible with single-AAV delivery, collectively offer compatibility with protospacer-adjacent motifs for editing approximately 82% of the adenines in the human genome. ABEs encoded within single AAVs will facilitate research and therapeutic applications of base editing by simplifying AAV production and characterization, and by reducing the dose required for the desired level of editing.
Topics: Animals; Humans; Mice; Dependovirus; Proprotein Convertase 9; Gene Editing; Adenine; Angiopoietin-like Proteins
PubMed: 35902773
DOI: 10.1038/s41551-022-00911-4 -
EMBO Molecular Medicine May 2022Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we...
Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock-in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV-based strategy carrying intein-split-dCas9 in combination with activators (AAV-dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single-cell transcriptome analysis of reprogrammed neurons in vivo. These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6-OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV-dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease.
Topics: Animals; Astrocytes; Corpus Striatum; Dopamine; Dopaminergic Neurons; GABAergic Neurons; Mice; Parkinson Disease
PubMed: 35373464
DOI: 10.15252/emmm.202114797 -
Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.Science Advances Apr 2021Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor...
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.
Topics: Animals; CRISPR-Cas Systems; Dystrophin; Exons; Gene Editing; Humans; Induced Pluripotent Stem Cells; Mice; Muscular Dystrophy, Duchenne; Sequence Deletion
PubMed: 33931459
DOI: 10.1126/sciadv.abg4910