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Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae

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Department of Biology and Biological Engineering, Chalmers University of Technology, SE412 96 Gothenburg, Sweden
Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE412 96 Gothenburg, Sweden
§ Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK2800 Kgs. Lyngby, Denmark
Cite this: ACS Synth. Biol. 2018, 7, 1, 10–15
Publication Date (Web):November 21, 2017
Copyright © 2017 American Chemical Society

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    Clustered regularly interspaced short palindromic repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made toward developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome editing and gene interference applications in S. cerevisiae. In regards to genome editing, we performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out of 24 colonies tested. Then, we used this system to efficiently transcriptionally regulate the three genes, OLE1, HMG1 and ACS1. Thus, we demonstrate that multiplexed genome editing and gene regulation can be performed in a fast and effective manner using Csy4.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00259.

    • Figures S1–S5; Tables S1–S6 (PDF)

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