Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 1 de 1
Filtrar
Más filtros

Banco de datos
Tipo del documento
Asunto de la revista
País de afiliación
Intervalo de año de publicación
1.
PLoS Biol ; 16(12): e2005595, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30540740

RESUMEN

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.


Asunto(s)
Reparación del ADN/fisiología , Heterocromatina/genética , Heterocromatina/fisiología , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Endonucleasas/metabolismo , Edición Génica/métodos , Genoma , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/fisiología , Mutagénesis Insercional , Mutágenos , Mutación/genética , Reparación del ADN por Recombinación/fisiología , Eliminación de Secuencia
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA