Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Growth, Reproductive Condition, And Digestive Tubule Atrophy Of Pacific Oyster Crassostrea Gigas In Gamakman Bay Off The Southern Coast Of Korea

Spat of Pacific oysters (Crassostrea gigas) were collected from Gamakman Bay, Korea, and raised in a spat hardening facility located in the low intertidal zone of the bay for a hardening/stunting period of 10 mo. Seasonal changes in growth, reproductive condition, and digestive tubule atrophy (DTA) of these hardened/stunted oysters were monitored for more than a year after transplanting to a suspended longline system in a grow-out area in the bay. After transplantation, the hardened/stunted oysters showed a logarithmic increase in shell size for the first 4 mo, from June to October, and growth remained stable from late fall to early spring. During the 12 mo of the grow-out, the shell size of the hardened/stunted oysters increased from 15.4-74.2 mm, and tissue weight increased from 0.49-12.85 g. Histological analysis revealed that gametogenesis of hardened/stunted oysters commenced as early as February when water temperature remained at 10 degrees C, and spawning occurred from July to September when water temperature reached 25-27 degrees C. DTA assessed from histological analysis was higher from September to February, when the chlorophyll a level in the bay was lower. These data suggest that seasonal fluctuations in water temperature and food availability in the water column are the 2 main environmental parameters governing reproduction and growth of oyster in Gamakman Bay, and DTA could be a useful biomarker for monitoring the nutritional condition of oysters

Gate-tuned normal and superconducting transport at the surface of a topological insulator

Three-dimensional topological insulators are characterized by the presence of a bandgap in their bulk and gapless Dirac fermions at their surfaces. New physical phenomena originating from the presence of the Dirac fermions are predicted to occur, and to be experimentally accessible via transport measurements in suitably designed electronic devices. Here we study transport through superconducting junctions fabricated on thin Bi2Se3 single crystals, equipped with a gate electrode. In the presence of perpendicular magnetic field B, sweeping the gate voltage enables us to observe the filling of the Dirac fermion Landau levels, whose character evolves continuously from electron- to hole-like. When B=0, a supercurrent appears, whose magnitude can be gate tuned, and is minimum at the charge neutrality point determined from the Landau level filling. Our results demonstrate how gated nano-electronic devices give control over normal and superconducting transport of Dirac fermions at an individual surface of a three-dimensional topological insulator.Comment: 28 pages, 5 figure

Atomically dispersed Pt-N-4 sites as efficient and selective electrocatalysts for the chlorine evolution reaction

Chlorine evolution reaction (CER) is a critical anode reaction in chlor-alkali electrolysis. Although precious metal-based mixed metal oxides (MMOs) have been widely used as CER catalysts, they suffer from the concomitant generation of oxygen during the CER. Herein, we demonstrate that atomically dispersed Pt-N-4 sites doped on a carbon nanotube (Pt-1/CNT) can catalyse the CER with excellent activity and selectivity. The Pt-1/CNT catalyst shows superior CER activity to a Pt nanoparticle-based catalyst and a commercial Ru/Ir-based MMO catalyst. Notably, Pt-1/CNT exhibits near 100% CER selectivity even in acidic media, with low Cl- concentrations (0.1M), as well as in neutral media, whereas the MMO catalyst shows substantially lower CER selectivity. In situ electrochemical X-ray absorption spectroscopy reveals the direct adsorption of Cl- on Pt-N-4 sites during the CER. Density functional theory calculations suggest the PtN4C12 site as the most plausible active site structure for the CER

Crystal Structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3 ' flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a 5 ' end binding pocket that hosts the 5 ' nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3 ' flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5 ' flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3 ' flap DNA substrates with 5 ' nicked ends.X11119Ysciescopu

A topological Dirac insulator in a quantum spin Hall phase : Experimental observation of first strong topological insulator

When electrons are subject to a large external magnetic field, the conventional charge quantum Hall effect \cite{Klitzing,Tsui} dictates that an electronic excitation gap is generated in the sample bulk, but metallic conduction is permitted at the boundary. Recent theoretical models suggest that certain bulk insulators with large spin-orbit interactions may also naturally support conducting topological boundary states in the extreme quantum limit, which opens up the possibility for studying unusual quantum Hall-like phenomena in zero external magnetic field. Bulk Bi$_{1-x}$Sb$_x$ single crystals are expected to be prime candidates for one such unusual Hall phase of matter known as the topological insulator. The hallmark of a topological insulator is the existence of metallic surface states that are higher dimensional analogues of the edge states that characterize a spin Hall insulator. In addition to its interesting boundary states, the bulk of Bi$_{1-x}$Sb$_x$ is predicted to exhibit three-dimensional Dirac particles, another topic of heightened current interest. Here, using incident-photon-energy-modulated (IPEM-ARPES), we report the first direct observation of massive Dirac particles in the bulk of Bi$_{0.9}$Sb$_{0.1}$, locate the Kramers' points at the sample's boundary and provide a comprehensive mapping of the topological Dirac insulator's gapless surface modes. These findings taken together suggest that the observed surface state on the boundary of the bulk insulator is a realization of the much sought exotic "topological metal". They also suggest that this material has potential application in developing next-generation quantum computing devices.Comment: 16 pages, 3 Figures. Submitted to NATURE on 25th November(2007

Ultra-low carrier concentration and surface dominant transport in Sb-doped Bi2Se3 topological insulator nanoribbons

A topological insulator is a new state of matter, possessing gapless spin-locking surface states across the bulk band gap which has created new opportunities from novel electronics to energy conversion. However, the large concentration of bulk residual carriers has been a major challenge for revealing the property of the topological surface state via electron transport measurement. Here we report surface state dominated transport in Sb-doped Bi2Se3 nanoribbons with very low bulk electron concentrations. In the nanoribbons with sub-10nm thickness protected by a ZnO layer, we demonstrate complete control of their top and bottom surfaces near the Dirac point, achieving the lowest carrier concentration of 2x10^11/cm2 reported in three-dimensional (3D) topological insulators. The Sb-doped Bi2Se3 nanostructures provide an attractive materials platform to study fundamental physics in topological insulators, as well as future applications.Comment: 5 pages, 4 figures, 1 tabl

The signalling channel of Central Bank interventions:modelling the Yen/US dollar exchange rate

This paper presents a theoretical framework analysing the signalling channel of exchange rate interventions as an informational trigger. We develop an implicit target zone framework with learning in order to model the signalling channel. The theoretical premise of the model is that interventions convey signals that communicate information about the exchange rate objectives of the central bank. The model is used to analyse the impact of Japanese FX interventions during the period 1999--2011 on the yen/US dollar dynamics

Josephson supercurrent through a topological insulator surface state

Topological insulators are characterized by an insulating bulk with a finite band gap and conducting edge or surface states, where charge carriers are protected against backscattering. These states give rise to the quantum spin Hall effect without an external magnetic field, where electrons with opposite spins have opposite momentum at a given edge. The surface energy spectrum of a threedimensional topological insulator is made up by an odd number of Dirac cones with the spin locked to the momentum. The long-sought yet elusive Majorana fermion is predicted to arise from a combination of a superconductor and a topological insulator. An essential step in the hunt for this emergent particle is the unequivocal observation of supercurrent in a topological phase. Here, we present the first measurement of a Josephson supercurrent through a topological insulator. Direct evidence for Josephson supercurrents in superconductor (Nb) - topological insulator (Bi2Te3) - superconductor e-beam fabricated junctions is provided by the observation of clear Shapiro steps under microwave irradiation, and a Fraunhofer-type dependence of the critical current on magnetic field. The dependence of the critical current on temperature and length shows that the junctions are in the ballistic limit. Shubnikov-de Haas oscillations in magnetic fields up to 30 T reveal a topologically non-trivial two-dimensional surface state. We argue that the ballistic Josephson current is hosted by this surface state despite the fact that the normal state transport is dominated by diffusive bulk conductivity. The lateral Nb-Bi2Te3-Nb junctions hence provide prospects for the realization of devices supporting Majorana fermions