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

CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid. Using CRISPR/Cas9 technology and Golden Gate Assembly, a toolkit including 147 plasmids and 137 promoters enables chromosomal integration of markerless DNA and easy assembly of new gRNAs, effectively proven in a novel Y. lipolytica production strain.

Automated summary

CRISPR/Cas9 technology is revolutionizing microbial cell engineering by enabling marker-free chromosomal integration of DNA. In this study, improvements were made to current Cas9 toolkits to make CRISPR technologies more accessible and easy to implement. These improvements include the ability to switch between marker-free and marker-based integration constructs, redirect multigene integration cassettes to alternative genomic loci, and assemble guide RNA sequences rapidly in vivo. These methodologies were combined with well-established technologies to create a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. The YaliCraft toolkit for Yarrowia lipolytica was developed as a proof of concept, demonstrating the successful integration of markerless DNA and easy assembly of new gRNAs, resulting in the production of homogentisic acid.