(641c) Combinatorial Crispri Expedites Microbial Metabolic Engineering | AIChE

(641c) Combinatorial Crispri Expedites Microbial Metabolic Engineering


Cress, B. F. - Presenter, Rensselaer Polytechnic Institute
Farrell, K. K., Rensselaer Polytechnic Institute
Linhardt, R. J., Rensselaer Polytechnic Institute
Koffas, M., Rensselaer Polytechnic Institute
Metabolic engineering is entering a new era in which addressable synthetic transcription factors like RNA-guided, catalytically inactive CRISPR effector proteins (dCas9, dCpf1, etc.) are opening new possibilities for dynamic regulation and for rapid assessment of multiplexed genetic interventions to alter the metabolic landscape in both workhorse and previously intractable microbes. Toward this end, our group has focused on several CRISPRi-enabled metabolic engineering applications. Previously, we developed a modular assembly method for traditional restriction-ligation cloning of CRISPR array libraries for multiplex, combinatorial dCas9-mediated transcriptional repression. Importantly, we demonstrated for the first time that dCas9 can be utilized for metabolic engineering in

Expanding upon this work, we have developed a novel, streamlined cloning strategy to assemble natural type II-A CRISPR array libraries in a one-pot reaction, enabling simultaneous repression of all genetic target combinations from a defined set in multiple disparate strains over the span of only a few days. We showcase this method by improving production of two distinct classes of flavonoids (naringenin and cyanidin 3-O-glucoside) through repression of several novel knockdown targets aimed at co-opting distinct, endogenous co-substrate pools. This ability to rapidly implement combinatorial genetic interventions in a variety of host strains potentially obviates the role of cumbersome combinatorial gene deletions during hypothesis testing stages early in the strain development pipeline.

Furthermore, to facilitate extension of this assembly method toward construction of distinct CRISPR array types that are compatible with the rapidly expanding repertoire of Class 2 CRISPR effectors, we have developed a web-accessible algorithm that outputs a design strategy and list of DNA oligonucleotides required to assemble defined or combinatorial libraries of any natural CRISPR array type/class. We showcase the algorithm by constructing functional CRISPR array libraries for two distinct CRISPR effector types. These studies underscore the immense impact that RNA-programmed synthetic transcription factors will have on the fields of metabolic engineering and synthetic biology.