A Cas3-base editing tool for targetable in vivo mutagenesis

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Genetic diversity is crucial for protein engineering and metabolic pathway optimization.
• Existing random mutagenesis methods have limitations in targeting specific genomic regions.
• There is a need for tools enabling precise and large-scale in vivo DNA mutagenesis.

Purpose of the Study:

• To develop a novel tool for inducible and targetable in vivo mutagenesis of large genomic loci.
• To enable efficient engineering of metabolic pathways through confined mutagenesis.
• To overcome the limitations of current whole-genome or narrow-window mutagenesis approaches.

Main Methods:

• Development of CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system).
• Utilizing the Cas3 helicase fused to a cytidine deaminase for unwinding and mutating large DNA stretches.
• Applying CoMuTER for inducible and targetable mutagenesis of genomic loci up to 55 kilobases.

Main Results:

• CoMuTER achieved a 350-fold increase in mutation frequency within the target region compared to the rest of the genome.
• An average mutation rate of 0.3 mutations per kilobase was observed in the targeted loci.
• Demonstrated successful pathway optimization by doubling lycopene production in Saccharomyces cerevisiae after one mutagenesis round.

Conclusions:

• CoMuTER is an effective tool for generating targeted genetic diversity in large genomic regions.
• The system facilitates efficient metabolic pathway engineering and protein engineering applications.
• CoMuTER significantly advances the capabilities for in vivo mutagenesis and strain development.