Tadas Jakočiūnas, Lasse E. Pedersen, Alicia V. Lis, Michael K. Jensen, Jay D. Keasling

Metabolic Engineering Volume 48, July 2018, Pages 288-296

Abstract

Here we describe a method for robust directed evolution using mutagenesis of large sequence spaces in their genomic contexts. The method employs error-prone PCR and Cas9-mediated genome integration of mutant libraries of large-sized donor variants into single or multiple genomic sites with efficiencies reaching 98–99%. From sequencing of genome integrants, we determined that the mutation frequency along the donor fragments is maintained evenly and successfully integrated into the genomic target loci, indicating that there is no bias of mutational load towards the proximity of the double strand break. To validate the applicability of the method for directed evolution of metabolic gene products we engineered two essential enzymes in the mevalonate pathway of Saccharomyces cerevisiae with selected variants supporting up to 11-fold higher production of isoprenoids. Taken together, our method extends on existing CRISPR technologies by facilitating efficient mutagenesis of hundreds of nucleotides in cognate genomic contexts.

See https://www.sciencedirect.com/science/article/pii/S109671761830123X#bib21

Figure 1: Schematic overview of experimental setup of CasPER method. CasPER is based on generation of mutagenized linear DNA fragments through repetitive rounds of error-prone PCR (epPCR) and a standard yeast transformation together with gRNA expression plasmid for genome integration.