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Nat Med ; 25(2): 229-233, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30664785


Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Edição de Genes , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Animais , Linhagem Celular , Técnicas de Introdução de Genes , Humanos , Camundongos , Primatas , Reprodutibilidade dos Testes , Visão Ocular
Genome Biol ; 16: 257, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26596280


BACKGROUND: CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9. RESULTS: We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq. CONCLUSION: Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.

Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Endodesoxirribonucleases/genética , Técnicas de Transferência de Genes , Engenharia Genética , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Desoxirribonuclease I/genética , Dependovirus/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de RNA , Streptococcus pyogenes/genética
Adv Exp Med Biol ; 806: 331-40, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24952190


The zebrafish (Danio rerio) is frequently being used to investigate the genetics of human diseases as well as resulting pathologies. Ease of both forward and reverse genetic manipulation along with conservation of vertebrate organ systems and disease causing genes has made this system a popular model. Many techniques have been developed to manipulate the genome of zebrafish producing mutants in a vast array of genes. While genetic manipulation of zebrafish has progressed, proteomics have been under-utilized. This review highlights studies that have already been performed using proteomic techniques and as well as our initial proteomic work comparing changes to the proteome between the ascl1a-/- and WT intestine.

Genoma/fisiologia , Mucosa Intestinal/metabolismo , Espectrometria de Massas/métodos , Proteômica/métodos , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Humanos , Mutação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
Dev Biol ; 376(2): 171-86, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23353550


The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a-/- embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a-/- embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34hpf and again between 64 and 74hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a-/- embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a-/- embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a-/- embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal intestinal motility.

Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células Epiteliais/citologia , Intestinos/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Enterocromafins/citologia , Enterócitos/metabolismo , Motilidade Gastrointestinal , Regulação da Expressão Gênica no Desenvolvimento , Células Caliciformes/citologia , Modelos Biológicos , Modelos Genéticos , Mutação , Transdução de Sinais , Peixe-Zebra