title: CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli.
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-specific 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.
authors.affiliation: 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.
authors.forename: Brady F
authors.initials: BF
authors.lastname: Cress
authors.affiliation: 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.
authors.forename: Ö Duhan
authors.initials: ÖD
authors.lastname: Toparlak
authors.forename: Sanjay
authors.initials: S
authors.lastname: Guleria
authors.affiliation: 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.
authors.forename: Matthew
authors.initials: M
authors.lastname: Lebovich
authors.affiliation: 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.
authors.forename: Jessica T
authors.initials: JT
authors.lastname: Stieglitz
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Englaender
authors.affiliation: 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.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Jones
authors.affiliation: 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.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.affiliation: 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.
authors.forename: Mattheos A G
authors.initials: MA
authors.lastname: Koffas
mesh_terms: D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004187:Disaccharides / Q000378:metabolism; D015536:Down-Regulation; D063185:Epigenetic Repression; D004926:Escherichia coli / Q000235:genetics*; D044950:Flavanones / Q000096:biosynthesis; D060847:Metabolic Engineering; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
chemical_list: D004187:Disaccharides; D044950:Flavanones; C560363:heparosan; C005273:naringenin
keywords: CRISPR array assembly; CRISPR/dCas9; gene regulation; heparosan; metabolic engineering; naringenin