Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Abstract

Controlling the copy number of gene expression cassettes is an important strategy to engineer bacterial cells into high-efficiency biocatalysts. Current strategies mostly use plasmid vectors, but multicopy plasmids are often genetically unstable, and their copy numbers cannot be precisely controlled. The integration of expression cassettes into a bacterial chromosome has advantages, but iterative integration is laborious, and it is challenging to obtain a library with varied gene doses for phenotype characterization. Here, we demonstrated that multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) can be achieved by designing a crRNA to target multicopy loci or a crRNA array to target multiple loci in the <i>Escherichia coli</i> genome. Within 5 days without selection pressure, <i>E. coli</i> strains carrying cargos with successively increasing copy numbers (up to 10) were obtained. Recombinant MUCICAT <i>E. coli</i> containing genomic multicopy glucose dehydrogenase expression cassettes showed 2.6-fold increased expression of this important industrial enzyme compared to <i>E. coli</i> harboring the conventional protein-expressing plasmid pET24a. Successful extension of MUCICAT to <i>Tatumella citrea</i> further demonstrated that MUCICAT may be generally applied to many bacterial species.