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Related Experiment Video

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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
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A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae.

Amanda Reider Apel1,2, Leo d'Espaux1,2, Maren Wehrs1,2

  • 1DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.

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Summary

This study introduces a CRISPR/Cas9 toolkit for faster yeast synthetic biology. It streamlines metabolic engineering by addressing common challenges, leading to a 25-fold increase in taxadiene production.

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Area of Science:

  • Synthetic biology
  • Molecular and cell biology
  • Biotechnology

Background:

  • Strain engineering in Saccharomyces cerevisiae for synthetic biology is often slow and labor-intensive.
  • Common challenges include selecting chromosomal integration sites, promoters, and managing protein localization and solubility.

Purpose of the Study:

  • To develop a cloning-free CRISPR/Cas9 toolkit to accelerate metabolic engineering in Saccharomyces cerevisiae.
  • To standardize and characterize genetic parts for improved yeast strain engineering.

Main Methods:

  • Employed CRISPR/Cas9 technology to create a toolkit with 23 Cas9-sgRNA plasmids, 37 promoters, and 10 protein tags.
  • Developed a web-based tool to automate DNA fragment generation for integration.
  • Standardized and characterized genetic parts for ease of use and reproducibility.

Main Results:

  • The toolkit addresses chromosomal integration, promoter selection, protein localization, and solubility.
  • A web-based tool simplifies the generation of DNA fragments for integration.
  • Optimized the expression of taxadiene synthase (TXS), identifying and resolving a solubility issue.

Conclusions:

  • The developed toolkit significantly enhances the efficiency of yeast metabolic engineering.
  • Resolved TXS solubility issues, resulting in a 25-fold improvement in taxadiene production.
  • This standardized and characterized toolkit offers a user-friendly methodology for genetic edits in yeast.