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Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae.

Tadas Jakočiūnas1, Ida Bonde1, Markus Herrgård1

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Summary

Multiplex CRISPR/Cas9 enables precise genome engineering in yeast, creating strains with over 41-fold higher mevalonate production. This efficient tool accelerates metabolic engineering for valuable compounds.

Keywords:
CRISPR/Cas9MevalonateMultiplex genome editingOff-target analysisYeast

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biotechnology

Background:

  • CRISPR/Cas9 is a versatile tool for targeted genome engineering.
  • Multiplex genome engineering allows simultaneous modification of multiple genetic loci.
  • Baker's yeast (Saccharomyces cerevisiae) is a key organism for industrial biotechnology.

Purpose of the Study:

  • To develop and validate a multiplex CRISPR/Cas9 system for engineering up to 5 genomic loci in Saccharomyces cerevisiae.
  • To assess the specificity and efficiency of the multiplex CRISPR/Cas9 system.
  • To identify yeast strains with enhanced mevalonate production through combinatorial gene disruption.

Main Methods:

  • Development of a multiplex CRISPR/Cas9 system for Saccharomyces cerevisiae.
  • Genome re-sequencing to evaluate off-target mutations in engineered strains.
  • Combinatorial disruption of up to five genes in the mevalonate pathway.
  • Quantification of mevalonate production in engineered yeast strains.

Main Results:

  • Successful engineering of up to 5 genomic loci in a single transformation step.
  • No detectable off-target mutations were identified via genome re-sequencing.
  • Identification of yeast strains exhibiting over 41-fold increased mevalonate titers compared to wild-type.
  • Demonstration of high specificity and efficiency of the multiplex CRISPR/Cas9 system.

Conclusions:

  • The developed multiplex CRISPR/Cas9 system is highly specific and efficient for yeast genome engineering.
  • This approach accelerates functional genomics and metabolic engineering for industrial applications.
  • Significant enhancement of mevalonate production is achievable through combinatorial gene disruption.