Multiplex Genome Engineering of Polyploid Industrial Yeast Strains Using an Optimized CRISPR/Cas9System

The Science

Researchers developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose‐fermenting, lactate‐producing industrial yeast strains, in which ALD6 , PHO13 , LEU2 , and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.

The Impact

  • Plasmids with higher copy numbers increased gRNA levels, resulting in higher genome editing efficiencies (up to 100% efficiency).
    • Up to 12 alleles were disrupted in a single step with 100% knock-out efficiency.
    • Facile engineering of industrial yeast strains for practical applications (i.e., biofuel production).
  • Optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast-based microbial cell factories.

References

Lian, J., Z. Bao, S. Hu, H. Zhao. 2017. “Engineered CRISPR/Cas9 system for Multiplex Genome Engineering of Polyploid Industrial Yeast Strains,” Biotechnology and Bioengineering 115(6), 1630–5. DOI:10.1002/bit.26569.