Journal article
CRISPR–Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Applied Metabolic Engineering, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Yeast Metabolic Engineering, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid.
Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR–Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously.
Our study provides a foundation for efficient engineering of industrial yeast cell factories.
| Language: | English |
|---|---|
| Publisher: | Elsevier |
| Year: | 2015 |
| Pages: | 13-22 |
| ISSN: | 22140301 |
| Types: | Journal article |
| DOI: | 10.1016/j.meteno.2015.03.001 |
| ORCIDs: | Stovicek, Vratislav , Borodina, Irina and Förster, Jochen |
Biorefineries CRISPR–Cas9 Chemical production Genome editing Industrial yeast SDG 7 - Affordable and Clean Energy
Biology (General) Biotechnology CRISPR–Cas9, clustered regularly interspaced short palindromic repeats–CRISPR-associated endonuclease 9 DSB, double strand break GOI, gene of interest HDR, homology-directed repair HR, homologous recombination NHEJ, non-homologous end joining PAM, protospacer adjacent motif PI, propidium iodide QH301-705.5 SNPs, single nucleotide polymorphisms TALENs, transcription activator-like effector nucleases TP248.13-248.65 USER, uracil-specific excision reaction ZFNs, zinc finger nucleases crRNA, CRISPR RNA gRNA, guide RNA tracrRNA, trans-activating RNA
