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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
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A synthetic RNA-mediated evolution system in yeast.

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  • 1Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.

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|June 9, 2021
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Summary

CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) enables rapid genetic adaptation by delivering evolving RNA donors directly into the genome. This method accelerates the discovery of new gene functions and traits in yeast.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Genetics

Background:

  • Laboratory evolution traditionally involves laborious, iterative cycles of mutagenesis and selection.
  • Existing methods for directed evolution are time-consuming or rely on natural adaptation processes.

Purpose of the Study:

  • To develop a novel, rapid method for in vivo directed evolution of genomic loci.
  • To enhance the efficiency and speed of discovering genetic adaptations for improved phenotypes.

Main Methods:

  • CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) utilizes evolving chimeric donor guide RNAs (gRNAs).
  • Error-prone T7 RNA polymerase generates continuously delivered RNA repair donors.
  • Cas9 or dCas9 guides the RNA donors for direct introduction into genomic targets.

Main Results:

  • CRAIDE successfully evolved novel functional variants of an auxotrophic marker gene in Saccharomyces cerevisiae.
  • The method conferred resistance to a toxic amino acid analogue, demonstrating its efficacy.
  • Achieved a mutation rate over 3,000-fold higher than the spontaneous native rate.

Conclusions:

  • CRAIDE represents a significant advancement in in vivo directed evolution.
  • Enables efficient genetic information transfer from evolving RNA templates to the genome.
  • Opens new avenues for rapid trait discovery and strain engineering without pre-programmed donors.