RESUMEN
The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Microbiota , Edición Génica/métodos , Humanos , Animales , Ratones , Microbiota/genética , Dependovirus/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Retina/metabolismo , Clostridiales/genética , Clostridiales/enzimología , Células HEK293 , Vectores Genéticos/metabolismo , Vectores Genéticos/genéticaRESUMEN
Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Inteínas , Retina/virología , Trans-Empalme/genética , Animales , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Organoides/ultraestructura , Organoides/virología , Fenotipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/virología , PorcinosRESUMEN
Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.