@inbook{93ae687ba7c14254ad32a43ff01bc370,
title = "Metabolic Engineering of Saccharomyces cerevisiae Using a Trifunctional CRISPR/Cas System for Simultaneous Gene Activation, Interference, and Deletion",
abstract = "Design and construction of an optimal microbial cell factory typically requires overexpression, knockdown, and knockout of multiple gene targets. In this chapter, we describe a combinatorial metabolic engineering strategy utilizing an orthogonal trifunctional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables multiplexed perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. To implement this system, three orthogonal Cas proteins were utilized: dLbCpf1 fused to a transcriptional activator, dSpCas9 fused to a transcriptional repressor, and SaCas9 for gene deletion. Deletion was accomplished by the introduction of a 28 bp frame-shift mutation using a homology donor on the guide RNA expression vector. This approach enables the application of metabolic engineering to systematically optimize phenotypes of interest through a combination of gain-, reduction-, and loss-of-function mutations. Finally, we describe the construction of the CRISPR-AID system and its application toward engineering an example phenotype, surface display of recombinant Trichoderma reesei endoglucanase II.",
keywords = "CRISPR, Genome engineering, Metabolic engineering, Saccharomyces cerevisiae, Synthetic biology",
author = "Carl Schultz and Jiazhang Lian and Huimin Zhao",
note = "Publisher Copyright: {\textcopyright} 2018 Elsevier Inc.",
year = "2018",
month = jan,
day = "1",
doi = "10.1016/bs.mie.2018.04.010",
language = "English (US)",
isbn = "9780128151488",
series = "Methods in Enzymology",
publisher = "Academic Press Inc.",
pages = "265--276",
editor = "Nigel Scrutton",
booktitle = "Methods in Enzymology",
address = "United States",
}