CRISPathBrick: Modular Combinatorial Assembly of Type II‑A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli Brady F. Cress, †,∥ O ̈ . Duhan Toparlak, †,∥ Sanjay Guleria, ∥ Matthew Lebovich, †,∥ Jessica T. Stieglitz, †,∥ Jacob A. Englaender, ‡,∥ J. Andrew Jones, †,∥ Robert J. Linhardt, †,‡,§,∥ and Mattheos A. G. Koffas* ,†,‡,∥ † Department of Chemical and Biological Engineering, ‡ Department of Biology, § Department of Chemistry and Chemical Biology, ∥ Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States * S Supporting Information ABSTRACT: Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems can be engineered to repress gene transcription at nearly any desired location in a sequence- speci fic manner, but there remain only a handful of applications described to date. In this work, we report development of a vector possessing a CRISPathBrick feature, enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in Escherichia coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of DNA parts, which can be utilized to generate a suite of complex phenotypes corresponding to an encoded genetic program. We show that CRISPathBrick can be used to tune expression of plasmid-based genes and repress chromosomal targets in probiotic, virulent, and commonly engineered E. coli strains. Furthermore, we describe development of pCRISPReporter, a fluorescent reporter plasmid utilized to quantify dCas9-mediated repression from endogenous promoters. Finally, we demonstrate that dCas9-mediated repression can be harnessed to assess the effect of downregulating both novel and computationally predicted metabolic engineering targets, improving the yield of a heterologous phytochemical through repression of endogenous genes. These tools provide a platform for rapid evaluation of multiplex metabolic engineering interventions. KEYWORDS: CRISPR/dCas9, metabolic engineering, gene regulation, naringenin, heparosan, CRISPR array assembly S elective and tunable perturbation of gene expression is a fundamental enabling technology in the fields of systems biology and synthetic biology, allowing the design of intricate synthetic circuits and the interrogation of complex natural biological systems. Until recently, however, there has been a paucity of tools to dynamically regulate transcription at the DNA level in a rapid, predictable, and specific manner. In the past, natural DNA-binding proteins have been harnessed by targeting to their cognate protein-binding sequences, artificially placed upstream or downstream of natural promoter sequences, to achieve transcriptional activation or repression; however, this method necessitates the addition of a static DNA element, or operator, near the promoter of interest. 1 This is especially problematic for regulation of endogenous genes since it requires genome engineering, a burdensome task for simultaneous manipulation of multiple targets. Conversely, programmable transcription factor (TF) proteins like zinc fingers and transcription activator like effectors (TALEs) have been utilized to target both natural and artificial DNA sequences for transcription modulation, but construction and selection of TFs are cumbersome processes that yield a TF capable of binding only a single target site. More elegant solutions for transcriptional regulation have been engineered using noncoding RNA (ncRNA) in a few noteworthy instances, 2−4 but, with the exception of a recent report, 5 these systems have suffered from limited predictability, design complexity, and a small dynamic range. 6 While translational repression can be achieved with other technologies like antisense RNA (asRNA), complex biological programs can benefit from, and might necessitate, multilevel interactions among RNA, DNA, and regulatory proteins, providing a strong argument for developing tools that can readily control transcription. One such tool, based on an engineered CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system, has recently been shown to achieve highly selective transcrip- tional modulation over a significant dynamic range. 7,8 Natural Received: January 26, 2015 Published: March 30, 2015 Research Article pubs.acs.org/synthbio © 2015 American Chemical Society 987 DOI: 10.1021/acssynbio.5b00012