Project Details
Projekt
Studying protein organization and dynamics of the Type I-Fv CRISPR-Cas system of Shewanella putrefaciens CN-32 at a high spatiotemporal resolution in living cells
Applicant
Professorin Dr. Ulrike Endesfelder
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Biophysics
Biophysics
Term
since 2018
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 405974843
The various CRISPR-Cas (Clustered regularly interspaced short palindromic repeats and CRISPR associated proteins) systems share some conserved features but are in general highly variable. While the organization of the Type II system and its signature protein Cas9 have already been studied in living cells using single-molecule methods, information on single-molecule (inter)dynamics of proteins of the Type I system stem from in vitro assays. There is, however, accumulating evidence that the interactions of CRISPR-Cas systems with their intracellular environment are much more complex than previously thought, going far beyond those of a monofunctional anti-viral defense mechanism. It is therefore becoming increasingly clear, that to fully understand their functionality, CRISPR-Cas systems cannot be studied in isolation.In this project, we will study the minimal CRISPR-Cas Type I-Fv system from Shewanella putrefaciens CN-32 in vivo using single-molecule localization microscopy (SMLM) and single-particle tracking (SPT). SMLM allows us to localize individual Cas molecules at a high spatiotemporal resolution and to observe their diffusion dynamics under different conditions and target affinities by SPT.In particular we aim to• characterize the kinetics of the Cascade Typ I-Fv complex in a spatiotemporal manner in living cells,• determine the kinetics and molecule interactions in PAM recognition and R-loop stabilization,• and to explore the significance of interactions between Cascade complexes and other proteins and molecular machineries.Furthermore, we aim to establish robust SPT and SMLM imaging in various less accommodating microorganisms, to be able to study CRISPR-Cas systems in their native host.
DFG Programme
Priority Programmes
