A3 - Resolving the mode of action of the CRISPR-Cas class I signature nuclease Cas3 inside cells with high spatiotemporal resolution

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CAS), e.g. nucleases, are best known for their unique capacity of genome engineering. The effector nuclease Cas9, common to all class II CRISPR/Cas systems, is well studied and has become a widely used tool. In contrast, class I systems, which are based on multi-protein complexes termed Cascade complexes that engage the nuclease Cas3, are less studied although they offer unique features. For example, Cas3 is a processive nuclease capable of efficiently degrading kilobase-long DNA segments. However, its molecular mechanism of action is not clear, e.g., i) there is in vitro evidence of different degradation modes of action of Cas3, ii) there are Cas3 variants showing bidirectional degradation including deletions downstream of the target site and the determinants for the segment length and direction(s) of degradation are unclear, and iii) there are indications of Cas3 functioning beyond antiviral defence. Considering the importance of the interplay of CRISPR/Cas with the cellular environment and the often highly transient nature of their interactions, we will study Cas3 using single-molecule localization microscopy (SMLM). With this approach, the dynamics of Cascade/Cas3 complex formation can be observed on the nanometer length and millisecond timescale in living cells. This allows deciphering which in vitro modes of action are physiologically relevant.