Probing the stability of the SpCas9-DNA complex after cleavage.

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.

Robust and fast post-processing of single-shot spin qubit detection events with a neural network.

Establishing low-error and fast detection methods for qubit readout is crucial for efficient quantum error correction. Here, we test neural networks to classify a collection of single-shot spin detection events, which are the readout signal of our qubit measurements. This readout signal contains a stochastic peak, for which a Bayesian inference filter including Gaussian noise is theoretically optimal. Hence, we benchmark our neural networks trained by various strategies versus this latter algorithm. Training of the network with 106 experimentally recorded single-shot readout traces does not improve the post-processing performance. A network trained by synthetically generated measurement traces performs similar in terms of the detection error and the post-processing speed compared to the Bayesian inference filter. This neural network turns out to be more robust to fluctuations in the signal offset, length and delay as well as in the signal-to-noise ratio. Notably, we find an increase of 7% in the visibility of the Rabi oscillation when we employ a network trained by synthetic readout traces combined with measured signal noise of our setup. Our contribution thus represents an example of the beneficial role which software and hardware implementation of neural networks may play in scalable spin qubit processor architectures.

Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions.

The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.