Targeted mutagenesis of multiple chromosomal regions in microbes

Directed evolution allows the effective engineering of proteins, biosynthetic pathways, and cellular functions. Traditional plasmid-based methods generally subject one or occasionally multiple genes-of-interest to mutagenesis, require time-consuming manual interventions, and the genes that are subjected to mutagenesis are outside of their native genomic context. Other methods mutagenize the whole genome unselectively which may distort the outcome. Recent recombineering- and CRISPR-based technologies radically change this field by allowing exceedingly high mutation rates at multiple, predefined loci in their native genomic context. In this review, we focus on recent technologies that potentially allow accelerated tunable mutagenesis at multiple genomic loci in the native genomic context of these target sequences. These technologies will be compared by four main criteria, including the scale of mutagenesis, portability to multiple microbial species, off-target mutagenesis, and cost-effectiveness. Finally, we discuss how these technical advances open new avenues in basic research and biotechnology.