Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system

Designing an optimal microbial cell factory often requires overexpression, knock-down, and knock-out of multiple gene targets. Unfortunately, such rewiring of cellular metabolism is often carried out sequentially and with low throughput. Here, we report a combinatorial metabolic engineering strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae . This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. We demonstrate the application of CRISPR-AID not only to increase the production of β-carotene by 3-fold in a single step, but also to achieve 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner. Metaboli engineering through gene overexpression, knock-down and knock-out is often carried out sequentially in a high labor, low-throughput manner. Here, the authors use CRISPR-mediated gene activation, interference and deletion to rapidly rewire S. cerevisiae metabolism in a single step.