Evolutionary Robust Many-Regulator Crispri Arrays for Large-Scale Reprogramming of Metabolism and Cellular Physiology | AIChE

Evolutionary Robust Many-Regulator Crispri Arrays for Large-Scale Reprogramming of Metabolism and Cellular Physiology

Authors 

Reis, A. - Presenter, Penn State University
Halper, S., Penn State Univ
Hossain, A., Pennsylvania State University
Salis, H., Pennsylvania State University
Vezeau, G., Pennsylvania State University
Clauer, P., Pennsylvania State University
We present a new CRISPR approach to simultaneously regulate the expression of up to 20 genes within an endogenous metabolic or signaling network, utilizing a newly developed toolbox of non-repetitive CRISPRi genetic parts designed for maximum evolutionary robustness. CRISPRi employs the deactivated version of Cas9 and single-guide RNA (sgRNA) regulators to bind to targeted DNA sites and repress transcription. Here, we show how to rationally design non-repetitive parts and optimally combine them together into Very Long sgRNA Arrays (VLSA) to implement complex metabolic redirections and cellular phenotypes. We demonstrated this technology by building and characterizing VLSAs for several applications: reprogramming metabolism to over-produce desired chemicals, creating auxotrophic GMO containment strains, and triggering increased cellular susceptibility to therapeutics. Notably, VLSAs can be synthesized as single DNA fragments and integrated into host genomes, efficiently and quickly, greatly accelerating the Synthetic Biology design-build-test cycle.