CRISPR
CRISPR interference
CRISPR interference allows us to quantitatively perturb single genes in bacteria. We constructed a CRISPRi library to perturb each gene that is involved in metabolism of E. coli and characterized growth of the library with FACS at the single-cell level (Beuter et al. 2018) and fitness by deep sequencing (Donati et al. 2020).
CRISPRi-knockdowns of enzymes enforce often very specific compensatory responses, indicating that cells can actively work against decreases of enzymes. From these responses, we infer the underlying regulatory mechanisms (Donati et al., 2020).
We also use CRISPRi to engineer better strains for biotechnological applications. For example, arginine producing E. coli performed much better when we used a knockdown of a transcription factor (ArgR) instead of the knockout (Sander et al 2019).
CRISPR genome editing
CRISPR allows us to make point mutations in bacterial genomes without markers or scars. For example, we used CRISPR to introduce point mutations into genes encoding allosteric enzymes in E. coli amino acid metabolism (Sander et al., 2019). These mutants showed decreases of enzyme levels, which in turn shows that allosteric feedback enforces enzyme overabundance.
Currently we use CRISPR to engineer temperature-sensitive proteins en masse. The motivation for this project is that temperature-sensitive enzymes can function as metabolic valves in industrial microbes (Schramm et al. 2020).