Adaptation of the CUT&Tag protocol to bacteria

CUT&Tag is a molecular biology method that combines protein A-Tn5 fusion-mediated targeted cleavage with DNA sequencing. This method identifies binding sites for DNA-associated proteins. While the method is developed for eukaryotes, a protocol is lacking for bacteria. This thesis aims to develop and optimize the CUT&Tag protocol for studying the epigenetic landscape and protein-DNA interactions in three bacterial species: Escherichia coli (E. coli), Mycobacterium smegmatis (M. smegmatis), and Staphylococcus aureus (S. aureus). The protocol was successfully applied to M. smegmatis where we demonstrate that Concanavalin-A magnetic beads are the best option to bind the bacteria and perform subsequent reactions in one tube. The sequencing will confirm our protocol optimization. For E. coli, binding to the bead was achieved, but permeabilization of the cell envelope posed challenges that we addressed with different techniques, including flow cytometry. Fluorescence imaging will be necessary to fully address this issue. For S. aureus, we assessed that Wheat Germ Agglutinin beads are the best binding option. In conclusion, we successfully achieved the first steps towards optimizing the CUT&Tag protocol across various bacterial strains. This will facilitate comprehensive bacterial genomics research and expedite pathogenic behavior comprehension for medical purposes.