RESUMO
The first clinical studies utilizing RNA-guided endonucleases (RGENs) to therapeutically edit RNA and DNA in cancer patients were recently published. These groundbreaking technological advances promise to revolutionize genetic therapy and, as I discuss, represent the culmination of decades of innovative work to engineer RGENs for such editing applications.
Assuntos
Edição de Genes/métodos , Edição de Genes/tendências , Edição de RNA/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , Endonucleases/metabolismo , Mutação , RNA/genética , Edição de RNA/fisiologia , RNA Catalítico/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The power of CRISPR is undeniable, but where is the field heading? Cell's April Pawluk caught up with Jia Chen, Weizhi Ji, and Prashant Mali to discuss the successes and challenges we can expect in the coming years. Annotated excerpts from this conversation are presented below, and the full conversation is available with the article online.
Assuntos
Edição de Genes/métodos , Edição de Genes/tendências , Terapia Genética/métodos , Terapia Genética/tendências , Genoma Humano , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , HumanosRESUMO
Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR-Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation.
Assuntos
Sistemas CRISPR-Cas , Tecnologia de Impulso Genético/métodos , Edição de Genes/métodos , Genética Populacional/métodos , RNA Guia de Cinetoplastídeos/genética , Alelos , Animais , Tecnologia de Impulso Genético/tendências , Edição de Genes/tendências , Humanos , Modelos Genéticos , Mutação , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Genome editing, which involves the precise manipulation of cellular DNA sequences to alter cell fates and organism traits, has the potential to both improve our understanding of human genetics and cure genetic disease. Here I discuss the scientific, technical and ethical aspects of using CRISPR (clustered regularly interspaced short palindromic repeats) technology for therapeutic applications in humans, focusing on specific examples that highlight both opportunities and challenges. Genome editing is-or will soon be-in the clinic for several diseases, with more applications under development. The rapid pace of the field demands active efforts to ensure that this breakthrough technology is used responsibly to treat, cure and prevent genetic disease.
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Anemia Falciforme/genética , Anemia Falciforme/terapia , Edição de Genes/métodos , Edição de Genes/tendências , Genoma Humano/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Sistemas CRISPR-Cas/genética , Edição de Genes/ética , Edição de Genes/normas , Mutação em Linhagem Germinativa/genética , Humanos , Especificidade de Órgãos/genética , Segurança do Paciente , Globinas beta/genéticaAssuntos
Sistemas CRISPR-Cas , Ensaios Clínicos como Assunto , Edição de Genes , Edição de RNA , RNA , Humanos , Ensaios Clínicos como Assunto/legislação & jurisprudência , Ensaios Clínicos como Assunto/normas , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Edição de Genes/tendências , RNA/genética , Edição de RNA/genéticaAssuntos
Doença de Alzheimer , Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Edição de Genes/tendências , Terapia Genética/métodos , Terapia Genética/tendências , MutaçãoAssuntos
Sistemas CRISPR-Cas , Vacinas Anticâncer , Edição de Genes , Neoplasias , Medicina de Precisão , Pesquisadores , Humanos , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Edição de Genes/tendências , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/genética , Medicina de Precisão/métodos , Medicina de Precisão/tendênciasRESUMO
Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs.
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Sistemas CRISPR-Cas/genética , Reparo do DNA/genética , Edição de Genes/tendências , Genoma Humano/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Recombinação Homóloga/genética , Humanos , Mutagênese Insercional/genética , Transdução de Sinais/genéticaRESUMO
IMPORTANCE: The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.
Assuntos
Brônquios , Dependovirus , Epitélio , Técnicas de Transferência de Genes , Vetores Genéticos , Transdução Genética , Humanos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , DNA , Epitélio/metabolismo , Epitélio/virologia , Técnicas de Transferência de Genes/tendências , Terapia Genética/métodos , Vetores Genéticos/genética , Brônquios/metabolismo , Brônquios/virologia , Transporte Ativo do Núcleo Celular , Edição de Genes/tendênciasAssuntos
Sistemas CRISPR-Cas , Camelus , Bovinos , Clonagem de Organismos , Edição de Genes , Ovinos , Animais , Bovinos/genética , Camelus/genética , Clonagem de Organismos/tendências , Clonagem de Organismos/veterinária , Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Edição de Genes/veterinária , Ovinos/genéticaAssuntos
Anemia Falciforme , Sistemas CRISPR-Cas , Edição de Genes , Talassemia beta , Anemia Falciforme/genética , Anemia Falciforme/terapia , Talassemia beta/genética , Talassemia beta/terapia , Sistemas CRISPR-Cas/genética , Edição de Genes/economia , Edição de Genes/legislação & jurisprudência , Edição de Genes/tendências , Reino Unido , HumanosRESUMO
Safe and effective heritable editing of the human genome is years away from the clinic because of formidable technical, statutory, regulatory, and societal challenges. In particular, we note the fledgling state of the science, the imperatives of editing efficiency, specificity, and uniformity, and the extant legal roadblock.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Genoma Humano/genética , Mutação em Linhagem Germinativa/genética , HumanosRESUMO
Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Sistemas CRISPR-Cas/genética , Citosina/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Doença de Stargardt/genética , Animais , Edição de Genes/tendências , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Mutação/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Doença de Stargardt/tratamento farmacológico , Doença de Stargardt/patologia , Linfócitos T/efeitos dos fármacos , Zigoto/efeitos dos fármacosRESUMO
The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.