RESUMEN
We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.
Asunto(s)
Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/análisis , Sitios de Unión , Proteínas Asociadas a CRISPR/genética , Citometría de Flujo , Colorantes Fluorescentes/análisis , Eliminación de Gen , Genes Reporteros , Ingeniería Genética/métodos , Vectores Genéticos , Genoma , Células HEK293 , Humanos , Microscopía Fluorescente , Mutagénesis , Mutación , Edición de ARN , Transcripción GenéticaRESUMEN
A key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonstrate that CRISPR regulatory devices can be layered to create functional cascaded circuits, which provide a valuable toolbox for engineering purposes.