A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering

Abstract CRISPR/Cas9 technology enables chromosomal integration of marker-free DNA, eliminating laborious marker recovery procedures. Nevertheless, assembling CRISPR/Cas9 editing systems is labour intensive and the vast majority of contemporary metabolic engineering projects do not involve this cutting-edge technology. We have identified key issues with current Cas9 toolkits and designed corresponding solutions. These include 1) A system to quickly switch between marker-free and marker-based integration constructs via Cre-expressing and standard Escherichia coli assembly strains. 2) The ability to redirect multigene integration donors into alternative genomic loci via Golden Gate – based exchange of homology arms. 3) A rapid, simple in-vivo method to re-encode guide sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies into a next-generation toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we characterized a library of 137 promoters and built a de novo Yarrowia lipolytica strain synthetizing 373.8 mg/L homogentisic acid.