title: Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9.
abstract: The progress in development of synthetic gene circuits has been hindered by the limited repertoire of available transcription factors. Recently, it has been greatly expanded using the CRISPR/Cas9 system. However, this system is limited by its imperfect DNA sequence specificity, leading to potential crosstalk with host genome or circuit components. Furthermore, CRISPR/Cas9-mediated gene regulation is context dependent, affecting the modularity of Cas9-based transcription factors. In this paper we address the problems of specificity and modularity by developing a computational approach for selecting Cas9/gRNA transcription factor/promoter pairs that are maximally orthogonal to each other as well as to the host genome and synthetic circuit components. We validate the method by designing and experimentally testing four orthogonal promoter/repressor pairs in the context of a strong promoter PL from phage lambda. We demonstrate that these promoters can be interfaced by constructing double and triple inverter circuits. To address the problem of modularity we propose and experimentally validate a scheme to predictably incorporate orthogonal CRISPR/Cas9 regulation into a large class of natural promoters.
authors.affiliation: BioCircuits Institute, ‡San Diego Center for Systems Biology, ¶Department of Bioengineering, and §Molecular Biology Section, Division of Biological Sciences, University of California San Diego , La Jolla, California 92093, United States.
authors.forename: Andriy
authors.initials: A
authors.lastname: Didovyk
authors.affiliation: BioCircuits Institute, ‡San Diego Center for Systems Biology, ¶Department of Bioengineering, and §Molecular Biology Section, Division of Biological Sciences, University of California San Diego , La Jolla, California 92093, United States.
authors.forename: Bartłomiej
authors.initials: B
authors.lastname: Borek
authors.affiliation: BioCircuits Institute, ‡San Diego Center for Systems Biology, ¶Department of Bioengineering, and §Molecular Biology Section, Division of Biological Sciences, University of California San Diego , La Jolla, California 92093, United States.
authors.forename: Jeff
authors.initials: J
authors.lastname: Hasty
authors.affiliation: BioCircuits Institute, ‡San Diego Center for Systems Biology, ¶Department of Bioengineering, and §Molecular Biology Section, Division of Biological Sciences, University of California San Diego , La Jolla, California 92093, United States.
authors.forename: Lev
authors.initials: L
authors.lastname: Tsimring
mesh_terms: D001665:Binding Sites; D064113:CRISPR-Cas Systems / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D024363:Transcription Initiation Site
chemical_list: D012097:Repressor Proteins
keywords: CRISPR/Cas9; orthogonal repressors; synthetic gene circuit engineering