Mechanism of assembly of an elongation-competent SARS-CoV-2 replication transcription complex

A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity...

Right place, Right Time: Modeling the search time and specificity of Cas9 and Argonaute

The past decade has witnessed a revolution in genome-engineering. Using CRISPR-Cas9 DNA sequences can be marked, detected and cleaved. Rewriting life’s instructions in such a fashion paves the way towards numerous scientific, agricultural and medical applications. Without proper quantfication of the associated risks we face the danger of...

Argonaute bypasses cellular obstacles without hindrance during target search

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses...

Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules

Due to their specificity, efficiency, and ease of programming, CRISPR-associated nucleases are popular tools for genome editing. On the genomic scale, these nucleases still show considerable off-target activity though, posing a serious obstacle to the development of therapies. Off targeting is often minimized by choosing especially high...

Why Argonaute is needed to make microRNA target search fast and reliable

MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss...

Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing...

Mean flow generation by Görtler Vortices in a rotating annulus with librating side walls

Longitudinal libration of the cylinder side walls of a rotating annulus in the supercritical regime induces a centrifugally unstable Stokes boundary layer which generates Görtler vortices only in a portion of a libration cycle. We show for the first time that these vortices propagate into the fluid bulk and generate an azimuthal mean flow which...

DNS of inertial wave attractors in a librating annulus with height-dependent gap width

Direct numerical simulations (DNS) of inertial wave attractors have been carried out in a librating Taylor-Couette system with broken mirror symmetry in the radial-axial cross-section. The inertial wave excitation mechanism and its localisation at the edges was clarified by applying boundary layer theory. Additional resonance peaks in the...

Mean Flow generation due to longitudinal librations of side-walls of a rotating annulus

Laboratory experiments with rotating annuli are reported that reveal a prograde jet, which is adjacent either to a (longitudinally) librating inner straight cylinder or to a librating inner truncated cone (frustum), whereas the outer cylindrical wall and bottom and top lids rotate with constant angular velocity. In the frustum case, the jet is...

Balanced ternary addition using a gated silicon nanowire

Ternary logic has the lowest cost of complexity, here, we demonstrate a CMOS hardware implementation of a ternary adder using a silicon metal-on-insulator single electron transistor. Gate dependent rectifying behavior of a single electron transistor (SET) results in a robust three-valued output as a function of the potential of the single...

Ternary logic implemented on a single dopant atom field effect silicon transistor

We provide an experimental proof of principle for a ternary multiplier realized in terms of the charge state of a single dopant atom embedded in a fin field effect transistor (Fin-FET). Robust reading of the logic output is made possible by using two channels to measure the current flowing through the device and the transconductance. A read out...