RNA-mediated in vivo Directed Evolution in Yeast.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Evolutionary Biology

Background:

• Directed evolution accelerates the discovery of biomolecules with enhanced traits by screening vast mutant libraries.
• Current directed evolution methods often involve laborious, repetitive steps for library preparation, propagation, and selection.
• Existing in vivo systems primarily focus on DNA evolution, with limited methods for RNA-based evolution.

Purpose of the Study:

• To develop a novel in vivo directed evolution system that facilitates the transfer of genetic information from RNA to DNA.
• To streamline the directed evolution process by reducing the need for extensive ex vivo manipulations.
• To enable the study of RNA's role in the evolution of DNA-based life.

Main Methods:

• Utilizing CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) in baker's yeast.
• Employing mutant T7 RNA polymerase to introduce targeted mutations into RNA donors.
• Directing the incorporation of mutated RNA sequences into DNA using CRISPR/Cas9 technology.

Main Results:

• Successful design and implementation of the CRAIDE protocol for RNA-to-DNA genetic information transfer.
• Demonstration of CRAIDE's capability to evolve RNA molecules with desired traits within a yeast system.
• Establishment of a streamlined in vivo directed evolution platform that bypasses traditional ex vivo steps.

Conclusions:

• CRAIDE represents a significant advancement in in vivo directed evolution, particularly for RNA-based evolution.
• The system simplifies the directed evolution workflow, making it more accessible and efficient.
• CRAIDE provides a unique platform for investigating fundamental evolutionary questions regarding RNA-DNA interactions.