2017 Annual Meeting
(641c) Combinatorial Crispri Expedites Microbial Metabolic Engineering
Authors
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.