A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

• 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.

Subject headings

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Bioinformatik och systembiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)

Publication and Content Type

art (subject category)

ref (subject category)