RESUMO
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.
Assuntos
Sistemas CRISPR-Cas/genética , RNA Guia de Cinetoplastídeos/análise , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Citometria de Fluxo , Corantes Fluorescentes/análise , Deleção de Genes , Genes Reporter , Engenharia Genética/métodos , Vetores Genéticos , Genoma , Células HEK293 , Humanos , Microscopia de Fluorescência , Mutagênese , Mutação , Edição de RNA , Transcrição GênicaRESUMO
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.
