Objective In the recent years a number of protein systems have been identified that recognize long (tens of base pairs) DNA sequences and allow flexible programmability of their target specificity. This promoted an enormous range of applications in genome engineering and synthetic biology. This project aims to decipher the mechanisms by which these proteins recognize their DNA targets in order to develop quantitative models/predictors for target recognition and to avoid off-target effects.To obtain detailed insight into the targeting mechanisms of different programmable systems in a “bottom-up manner”, cutting-edge single-molecule experiments, such as mechanical DNA twisting combined with single-molecule fluorescence detection will be employed. This will provide a fully quantitative characterization of the targeting process and insight into the mechanisms of allosteric regulation coupled to targeting. The quantitative data will allow to develop physics-based models of the target recognition process. In particular, we will focus on recognition through non-equilibrium, directional zipping along the target sequence – as recently revealed for CRISPR-Cas enzymes – as a promising unifying mechanism. To obtain precise targeting predictors our first-principle models will be tested and refined using high-throughput measurements on many different targets in parallel. Finally, the predictions will be used in order to understand target selection in live cells using single-molecule imaging.Within the project the following goals are defined: Goal 1: Quantitative understanding of target binding/degradation for CRISPR-Cas systemsGoal 2: Detailed mechanistic insight into the target recognition process by TALEs Goal 3: Development of highly parallelized measurements on different target sequences down to the single-molecule levelGoal 4: Target identification in the complex environment of live cells Fields of science natural sciencesbiological sciencessynthetic biologynatural sciencesbiological sciencesgeneticsDNAnatural sciencesbiological sciencesgeneticsgenomesnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Keywords single-molecule biophysics force torque spectroscopy random-walk model CRISPR-Cas DNA recognition Programme(s) H2020-EU.1.1. - EXCELLENT SCIENCE - European Research Council (ERC) Main Programme Topic(s) ERC-2016-COG - ERC Consolidator Grant Call for proposal ERC-2016-COG See other projects for this call Funding Scheme ERC-COG - Consolidator Grant Coordinator UNIVERSITAET LEIPZIG Net EU contribution € 2 000 000,00 Address Ritterstrasse 26 04109 Leipzig Germany See on map Region Sachsen Leipzig Leipzig Activity type Higher or Secondary Education Establishments Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Other funding € 0,00 Beneficiaries (1) Sort alphabetically Sort by Net EU contribution Expand all Collapse all UNIVERSITAET LEIPZIG Germany Net EU contribution € 2 000 000,00 Address Ritterstrasse 26 04109 Leipzig See on map Region Sachsen Leipzig Leipzig Activity type Higher or Secondary Education Establishments Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Other funding € 0,00
