A Cas3-base editing tool for targetable in vivo mutagenesis

Abstract 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

Btn2p sequesters prion amyloid filaments, curing the prion.

<p>Btn2p gathers filaments of Ure2p amyloid (the [URE3] prion) to a single site in the cell, possibly the endosome. If the prion has a low seed number then even the normal levels of Btn2p are sufficient to sequester nearly all of the seeds, so that daughter cells without seeds, and therefore cured of the prion, are often produced.</p

Protein conformation templating mechanism.

<p>The in-register parallel amyloid architecture naturally suggests a mechanism for transfer of conformation information from molecules in the filament to those joining the filament. H-bonding or hydrophobic favorable interactions among identical side chains require in-register alignment for the interactions. This directs the monomer joining the end of the filament to have its folds/turns at the same residues as previous molecules in the filament. Different prion variants have folds/turns at different locations, but each is faithfully propagated by this mechanism. Modified from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004584#ppat.1004584.ref006" target="_blank">6</a>].</p