Metabolic Engineering of Saccharomyces cerevisiae Using a Trifunctional CRISPR/Cas System for Simultaneous Gene Activation, Interference, and Deletion

Carl Schultz, Jiazhang Lian, Huimin Zhao

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsNigel Scrutton
PublisherAcademic Press Inc.
Pages265-276
Number of pages12
ISBN (Print)9780128151488
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Enzymology
Volume608
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • CRISPR
  • Genome engineering
  • Metabolic engineering
  • Saccharomyces cerevisiae
  • Synthetic biology

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Schultz, C., Lian, J., & Zhao, H. (2018). Metabolic Engineering of Saccharomyces cerevisiae Using a Trifunctional CRISPR/Cas System for Simultaneous Gene Activation, Interference, and Deletion. In N. Scrutton (Ed.), Methods in Enzymology (pp. 265-276). (Methods in Enzymology; Vol. 608). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.04.010