GENETIC TOOLS

Our laboratory is at the forefront of lactic acid bacteria genome editing. Since several projects in our laboratory focus on the gut symbiont Limosilactobacillus reuteri, we use this species as our model to develop new genome editing technologies.

Recombineering. We routinely apply single-stranded DNA recombineering (SSDR) to modify the bacterial chromosome. A short oligonucleotide is introduced in bacterial cells that temporarily express a recombinase protein. The recombinase helps to stabilize, and possibly protect, the oligonucleotide. The oligonucleotide serves as an Okazaki fragment, and is extended by DNA polymerase into a new strand. User-defined non-homologous bases in the oligonucleotide will result in targeted base changes in the chromosome.

CRISPR. In addition, we pioneered the application of CRISPR-Cas-assisted recombineering in lactic acid bacteria. CRISPR-Cas serves as a tool to enrich the recombinant genotype in a population. This opens up the exciting opportunity to identify low-efficiency mutagenesis events. For example, implementing CRISPR-Cas means we can identify up to 1kb chromosomal deletions generated by a single oligonucleotide. We also demonstrated we can modify a single codon to encode 19 additional amino acids. All mutants were generated by a single person within a couple of days.

Counterselection system. While SSDR and CRISPR-Cas are very powerful technologies, the efficacy by which these tools function vary per strain. Therefore, we developed a counterselection system that functions in many members of the family Lactobacillaceae. Unlike most counterselection systems, our platform does not require prior genome editing and can be combined with a liquid-based approach to generate insertions or deletions in a matter of days.

For more in-depth information on these genome editing technologies, we refer to these select publications.