Crispathbrick: Modular Combinatorial Assembly of Type II-a Crispr Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli | AIChE

Crispathbrick: Modular Combinatorial Assembly of Type II-a Crispr Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli

Authors 

Toparlak, D., Rensselaer Polytechnic Institute
Lebovich, M., Rensselaer Polytechnic Institute
Englaender, J. A., Rensselaer Polytechnic Institute

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 cloning procedure called “CRISPathBrick,” enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in E. coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of synthetic, user-defined DNA parts, which can be utilized to generate a suite of complex phenotypes. We demonstrate assembly and functionality of these arrays in E. coli by repressing genomic and plasmid-based reporter genes, as well as endogenous virulence factors and biosynthetic genes, using a single plasmid in multiple divergent strains. We show that significant repression is achieved in engineering hosts like BL21, K-12, and DH5alpha, in addition to wild-type probiotic strain Nissle 1917 and virulent strain K5. The value of addressable, orthogonal master transcriptional regulators like dCas9 cannot be overstated; thus, we have developed a two-plasmid toolkit for facile construction and characterization of CRISPR arrays. The first plasmid, pCRISPathBrick, possesses all components necessary to achieve repression of both heterologous and endogenous targets in E. coli, guided by the genetic program encoded in its readily expandable CRISPR array. The second plasmid, pCRISPReporter, is compatible with pCRISPathBrick and contains a transcriptionally-insulated multiple cloning site, where genomic promoters regions and genes of interest can be inserted to create a translational fusion with any desired reporter. With these two plasmids, we demonstrate facile characterization of synthetic spacers, quantified in terms of repression of the target protein-reporter fusion. Finally, we show that CRISPathBrick can be used to repress endogenous metabolic targets in E. coli, mediating up to 2.5-fold improvement in production of the heterologous plant natural product naringenin (from 7.6 mg/L to 18.9 mg/L). We believe that the tools developed here will be incredibly valuable for scientists in many disciplines, from systems and synthetic biology to metabolic engineering and the basic sciences.