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

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...

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Bibliographic Details
Published inACS synthetic biology Vol. 7; no. 1; pp. 10 - 15
Main Authors Ferreira, Raphael, Skrekas, Christos, Nielsen, Jens, David, Florian
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 19.01.2018
Subjects
Acyl Coenzyme A - deficiency
Acyl Coenzyme A - genetics
Bacterial Proteins - genetics
Coenzyme A Ligases - deficiency
Coenzyme A Ligases - genetics
CRISPR
CRISPR-Associated Proteins - genetics
CRISPR-Cas Systems - genetics
CRISPRi
Csy4
Endoribonucleases - genetics
gene editing
Gene Editing - methods
gene regulation
Metabolic Engineering
multiplexing
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Stearoyl-CoA Desaturase - genetics
Stearoyl-CoA Desaturase - metabolism
Transcription, Genetic
CRISPR
CRISPRi
multiplexing
gene regulation
Csy4
metabolic engineering
gene editing
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Summary: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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ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.7b00259