RESUMEN

Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

Asunto(s)

Sistemas CRISPR-Cas/genética , Clonación Molecular , Técnicas de Sustitución del Gen , Animales , Línea Celular , Femenino , Marcación de Gen , Genes Reporteros , Sitios Genéticos , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN