Clustered, Regularly-Interspaced Short Palindromic Repeats (CRISPR) and their associated enzyme (Cas9) are a widely used tools for mutagenesis like insertion, deletion and point mutations. In recent years the implementation of CRISPR-Cas9 systems in procaryotes has increased. It is an excellent tool for genome editing of known strains of Lactococcus lactis as NZ9000, but little research focuses on the editing of starter culture strains.

In this study, CRISPR-Cas9 will be established in L. lactis strains isolated from cheese manufacture. The aim is to create a CRISPR-Cas9 system that can achieve site specific mutagenesis of a cell-enveloped proteinase gene to investigate the effects on the bitter peptide content in dairy products.

Two strains of L. lactis isolated from cheese production were used. The plasmid systems for CRISPR-Cas9 mediated Gene mutagenesis were developed by combining bioinformatic methods, CRISPR-Cas9 programs and the results of whole genome sequencing.

The comparison of the different tools shows that not all systems already developed in L. lactis NZ9000 can be transferred to L. lactis starter cultures without problems. The nisin-induced systems can only be used by introducing the necessary genes via another vector. The applied L. lactis strains from starter cultures contain native plasmids. Therefore, a comparison of the origin of replication of the plasmids is necessary. Furthermore, it must be investigated whether the strains' own recombinase activity is sufficient to effectively utilize single plasmid systems without foreign recombinase. In addition, it must be investigated whether the already published single plasmid systems, which are mainly used for deletion, are suitable for a single base pair exchange if the repair template is integrated on the CRISPR-Cas9 vector.

In summary, plasmid systems designed in L. lactis NZ9000 cannot be easily transferred to L. lactis strains present in starter cultures. Further studies are needed to show whether a single plasmid system consisting of only one CRISPR-Cas9 plasmid is sufficient to perform site specific mutagenesis in L. lactis isolates.