A Cas3-base editing tool for targetable in vivo mutagenesis

The generation of genetic diversity via mutagenesis is routinely used for protein engineering and pathway optimization. Current technologies for random mutagenesis often target either the whole genome or relatively narrow windows. To bridge this gap, we developed CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system), a tool that allows inducible and targetable, in vivo mutagenesis of genomic loci of up to 55 kilobases. CoMuTER employs the targetable helicase Cas3, signature enzyme of the class 1 type I-E CRISPR-Cas system, fused to a cytidine deaminase to unwind and mutate large stretches of DNA at once, including complete metabolic pathways. The tool increases the number of mutations in the target region 350-fold compared to the rest of the genome, with an average of 0.3 mutations per kilobase. We demonstrate the suitability of CoMuTER for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.

Automated summary

CoMuTER is a new tool that utilizes the targetable helicase Cas3 and cytidine deaminase to induce mutations in large stretches of DNA, allowing for protein engineering and pathway optimization. It increases the number of mutations in the target region by 350-fold, with an average of 0.3 mutations per kilobase. We demonstrate its suitability for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.