Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9

• DOI: 10.1021/acssynbio.5b00147
• Publication Date: 2015-09-21T18:29:26Z
• Authors: Andriy Didovyk; Bartłomiej Borek; Jeff Hasty; Lev Tsimring
• Keywords: Synthetic Biology; Trans-activating crRNA; CRISPR interference; Transcription; Modularity

The progress in development of synthetic gene circuits has been hindered by the limited repertoire available transcription factors. Recently, it greatly expanded using CRISPR/Cas9 system. However, this system is its imperfect DNA sequence specificity, leading to potential crosstalk with host genome or circuit components. Furthermore, CRISPR/Cas9-mediated regulation context dependent, affecting modularity Cas9-based In paper we address problems specificity and developing a computational approach for selecting Cas9/gRNA factor/promoter pairs that are maximally orthogonal each other as well We validate method designing experimentally testing four promoter/repressor strong promoter PL from phage lambda. demonstrate these promoters can be interfaced constructing double triple inverter circuits. To problem propose scheme predictably incorporate into large class natural promoters.