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The discovery1,2and the pioneering applications3of CRISPR-Cas effector complexes have provided powerful gene-editing tools. The effector complexes are guided to the targeted genomic locus by the complementarity of their CRISPR RNA (crRNA)4,5. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base-pairing between crRNA and the DNA target strand resulting in the formation of an R-loop structure5,6. Full R-loop formation is the prerequisite for the subsequent DNA cleavage. While the CRISPR-Cas technology is easy to use, efficient and highly versatile, therapeutic applications are hampered by the off-target effects due to the recognition of unintended sequences with multiple mismatches7. This process is still poorly understood on a mechanistic level8,9. Particularly, the lack of insight into the energetics and dynamics of the R-loop formation hinders a direct modelling of the R-loop formation for off-target prediction.Here we set up ultrafast DNA unwinding experiments based on plasmonic DNA nanorotors to follow the R-loop formation by the Cascade effector complex in real time, close to base pair resolution. We directly resolve a weak global downhill bias of the energy landscape of the forming R-loop followed by a steep uphill bias for the final base pairs. We furthermore show a modulation of the landscape by base flips and mismatches. These data provide that Cascade-mediated R-loop formation occurs on short time scales in single base pair steps of sub-millisecond duration, but on longer time scales in six–base pair intermediate steps in agreement with the structural periodicity of the crRNA-DNA hybrid. We expect that the knowledge about the energy landscapes of R-loop formation of CRISPR-Cas effector complexes will pave the way for a detailed understanding and prediction of off-target recognition10.

Cleavage of bacteriophage DNA by the Type III Restriction-Modification enzymes requires long-range interaction between DNA sites. This is facilitated by 1-dimensional diffusion (“DNA sliding”) initiated by ATP hydrolysis catalysed by a superfamily 2 helicase-like ATPase. Here we combined ultrafast twist measurements based on plasmonic DNA origami nano-rotors with stopped-flow fluorescence and gel-based assays to examine the role(s) for ATP hydrolysis. Our data shows that the helicase-like domain plays multiple roles. Firstly, stabilising initial DNA-interactions alongside the methyltransferase subunits. Secondly, causing environmental changes in the flipped adenine base following hydrolysis of the first ATP. And thirdly, remodelling nucleoprotein interactions via constrained translocation of a ~5-20 bp dsDNA loop. Initiation of DNA sliding requires 10-15 bp DNA downstream of the motor, corresponding to the site of nuclease domain binding. Our data unifies previous contradictory communication models for Type III enzymes.

CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors first have to interrogate the entire genome for the presence of a matching sequence. Matching is achieved by base-pairing between the crRNA of the complexes and the DNA target strand such that an R-loop is formed. R-loop formation starts at a specific PAM motif and progresses reversibly in single base-pair steps until mismatches stop further progression or until the full target is recognized and destroyed. The reversible nature of this process entails that even a fully matching target should only become recognized with a low probability per target encounter. The details of this process, which directly affect the effectiveness of the target search, remain unresolved.Here we dissect the target search process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the low target recognition probabilities and show that they increase with increasing negative supercoiling. Furthermore, we demonstrate that Cascade uses a combination of three-dimensional and limited one-dimensional diffusion along the DNA contour for its target search. The latter allows for rapidly scanning the PAM sequences in a given region and, importantly, significantly increasing the overall efficiency of the target search by repeatedly revisiting the sites. Overall we show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

The development of the DNA origami technique has directly inspired the idea of using three-dimensional DNA cages for the encapsulation and targeted delivery of drug or cargo molecules. The cages would be filled with molecules that would be released at a site of interest upon cage opening triggered by an external stimulus. Though different cage variants have been developed, efficient loading of DNA cages with freely-diffusing cargo molecules that are not attached to the DNA nanostructure and their efficient retention within the cages has not been presented. Here we address these challenges using DNA origami nanotubes formed by a double-layer of DNA helices that can be sealed with tight DNA lids at their ends. In a first step we attach DNA-conjugated cargo proteins to complementary target strands inside the DNA tubes. After tube sealing, the cargo molecules are released inside the cavity using toehold-mediated strand displacement by externally added invader strands. We show that DNA invaders are rapidly entering the cages through their DNA walls. Retention of ∼70 kDa protein cargo molecules inside the cages was, however, poor. Guided by coarse-grained simulations of the DNA cage dynamics, a tighter sealing of the DNA tubes was developed which greatly reduced the undesired escape of cargo proteins. These improved DNA nanocages allow for efficient encapsulation of medium-sized cargo molecules while remaining accessible to small molecules that can be used to trigger reactions, including a controlled release of the cargo

Here we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand-displacement reactions. The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables the strand displacement reaction. The kinetics of the blocker enzymatic degradation thus controls the time at which the strand displacement reaction starts. By varying the concentration of the blocker strand and the concentration of the enzyme we show that we can finely tune and modulate the delayed onset of the strand displacement reaction. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed strand displacement reactions using RNase H and the two DNA repair enzymes Fpg and UDG and corresponding blockers. The achieved temporal delay can be programmed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show that the delayed strand displacement reactions can be coupled to downstream processes and used to control the onset of ligand release from a DNA nanodevice as well as protein inhibition by a DNA aptamer.

Here we show a general approach to achieve dissipative control over toehold-mediated strand-displacement, the most widely employed reaction in the field of DNA nanotechnology. The approach relies on rationally re-engineering the classic strand displacement reaction such that the high-energy invader strand (fuel) is converted into a low-energy waste product through an energy-dissipating reaction allowing the spontaneous return to the original state over time. We show that such dissipative control over the toehold-mediated strand displacement process is reversible (up to 10 cycles), highly controllable and enables unique temporal activation of DNA systems. We show here two possible applications of this strategy: the transient labelling of DNA structures and the additional temporal control of cascade reactions.

Nachdenken hilft! Auch beim Gespräch mit der nachfolgenden Generation über die Bedeutung der Vergangenheit und die Einordung des Erlebten. Ralf Seidel war über 20 Jahre Ärztlicher Direktor der Landesklinik Mönchengladbach. Bei Beginn seiner Arbeit als Psychiater hatte die Sachverständigenkommission für die Psychiatrie-Enquete gerade ihren ersten Zwischenbericht an den Bundestag übergeben. Ralf Seidel hat die Psychiatriereform in Deutschland nicht nur miterlebt, sondern auch mitgeprägt. Seine Interessen gingen dabei immer über sein Fach hinaus. Ralf Seidel hat nicht nur Medizin studiert, sondern auch Philosophie; er ist der Kunst sehr verbunden und weit gereist. Ein ungewöhnliches Arztleben zwischen Aufbruch, Reform und Reflexion.

Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G2-M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability., Science Advances, 8 (5), ISSN:2375-2548