Understanding and engineering DNA targeting by CRISPR-Cas effectors using ultra-fast DNA twist measurements

• Applicant: Professor Dr. Ralf Seidel
• Subject Area: Biophysics
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 568641279

CRISPR-Cas effector complexes, such as Cas9, have revolutionized genome engineering technologies in recent years. The challenge in these applications lies in achieving both highly efficient and highly specific DNA cleavage. This research project aims to resolve, with near base-pair resolution, the dynamics of target recognition by Cas9 and Cas12 effector complexes to establish precise kinetic models that can predict targeting properties. We will employ recently developed ultra-fast, high-resolution DNA untwisting measurements based on single DNA origami nanorotors. This methodology will enable real-time tracking of R-loop expansion and shortening by the effectors, allowing direct resolution of critical kinetic intermediates and the free energy landscape of R-loop formation. The study will systematically quantify how intermediate states and free energy landscapes are modulated by different effector variants and RNA guide sequences. This shall provide a comprehensive understanding of how protein- and RNA sequence thermodynamics influence the targeting properties. The obtained experimental data then be used to parameterize kinetic models that accurately predict the efficiency and specificity of target binding and DNA cleavage by various CRISPR-Cas effectors. We will validate these models by comparing their performance against available high-throughput data and previous models that have not considered the thermodynamic contributions to the targeting process.

• DFG Programme: Research Grants