Optimization of a Genome Editing System for Lactococci Based on a CRISPR-Associated Transposon

Abstract Lactococcus lactis is a microbial species widely used in the dairy industry as a component of starter cultures; it is also employed as a producer of recombinant proteins and biologically active metabolites, as well as in other biotechnological processes. However, genomic editing of these bacteria aimed at conferring them the desired technological properties faces significant challenges due to the lack of convenient tools for rapid and efficient gene knockout and for insertion of large genetic cassettes (several kb long) into the chromosome. Previously, we proposed an adaptation of a system based on a CRISPR-Cas-directed transposon for use in L. lactis. In this work, we improved the previously developed tool by designing a single-plasmid variant instead of the two-plasmid one and by adapting the tetracycline resistance gene as an additional selective marker in L. lactis. The resulting system significantly simplifies and accelerates the delivery of the required genetic material into the chromosome and allows for sequential modification of three different sites using readily available resistance markers (tetracycline, chloramphenicol, and erythromycin resistance genes), which can be later deleted in a single step.