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dc.contributor.authorCress, Brady F.
dc.contributor.authorToparlak, O. Duhan
dc.contributor.authorGuleria, Sanjay
dc.contributor.authorLebovich, Matthew
dc.contributor.authorStieglitz, Jessica T.
dc.contributor.authorEnglaender, Jacob A.
dc.contributor.authorJones, J. Andrew
dc.contributor.authorLinhardt, Robert J.
dc.contributor.authorKoffas, Mattheos A.G.
dc.date2015
dc.date.accessioned2022-06-27T16:04:33Z
dc.date.available2022-06-27T16:04:33Z
dc.date.issued2015-03-30
dc.identifier.citationCRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli, B. F. Cress, Ö. D. Toparlak, S. Guleria, M. Lebovich, J. T. Stieglitz, J. A. Englaender, J. A. Jones, R. J. Linhardt, M. A. G. Koffas, ACS Synthetic Biology, 4, 987–1000, 2015.
dc.identifier.issn21615063
dc.identifier.urihttps://doi.org/10.1021/acssynbio.5b00012
dc.identifier.urihttps://hdl.handle.net/20.500.13015/5672
dc.descriptionACS Synthetic Biology, 4, 987–1000
dc.descriptionNote : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
dc.description.abstractProgrammable 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.
dc.description.sponsorshipNational Science Foundation
dc.description.urihttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1021/acssynbio.5b00012
dc.languageen_US
dc.language.isoENG
dc.relation.ispartofThe Linhardt Research Labs Online Collection
dc.relation.ispartofRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofACS Synthetic Biology
dc.relation.urihttps://harc.rpi.edu/
dc.subjectBiology
dc.subjectChemistry and chemical biology
dc.subjectChemical and biological engineering
dc.subjectBiomedical engineering
dc.titleCRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli
dc.typeArticle
dcterms.accessRightshttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1021/acssynbio.5b00012
dcterms.isPartOfJournal
dcterms.isVersionOfhttps://doi.org/10.1021/acssynbio.5b00012
dc.rights.holderIn Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). https://rightsstatements.org/page/InC/1.0/
dc.creator.identifierhttps://orcid.org/0000-0003-2219-5833
dc.relation.departmentThe Linhardt Research Labs.
dc.relation.departmentThe Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
rpi.description.pages987-1000
rpi.description.volume4


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