A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12
Journal article, 2020

Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to similar to 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains.

Author

Elena Cámara

Chalmers, Biology and Biological Engineering, Industrial Biotechnology

Ibai Lenitz Etxaburu

Chalmers, Biology and Biological Engineering, Industrial Biotechnology

Yvonne Nygård

Chalmers, Biology and Biological Engineering, Industrial Biotechnology

Scientific Reports

2045-2322 (ISSN)

Vol. 10 1 14605

Novel methods for accelerating the development of more inhibitor tolerant strains for cellulosic ethanol production

Swedish Energy Agency, 2017-05-01 -- 2019-04-30.

Subject Categories

Biochemistry and Molecular Biology

Microbiology

Bioinformatics and Systems Biology

Areas of Advance

Energy

DOI

10.1038/s41598-020-71648-w

PubMed

32884066

More information

Latest update

10/23/2020