In vivo genome editing restores haemostasis in a mouse model of haemophilia

Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.     

Grains and grain boundaries in single-layer graphene atomic patchwork quilts

The properties of polycrystalline materials are often dominated by the size of their grains and by the atomic structure of their grain boundaries. These effects should be especially pronounced in two-dimensional materials, where even a line defect can divide and disrupt a crystal. These issues take on practical significance in graphene, which is a hexagonal, two-dimensional crystal of carbon atoms. Single-atom-thick graphene sheets can now be produced by chemical vapour deposition on scales of up to metres, making their polycrystallinity almost unavoidable. Theoretically, graphene grain boundaries are predicted to have distinct electronic, magnetic, chemical and mechanical properties that strongly depend on their atomic arrangement. Yet because of the five-order-of-magnitude size difference between grains and the atoms at grain boundaries, few experiments have fully explored the graphene grain structure. Here we use a combination of old and new transmission electron microscopy techniques to bridge these length scales. Using atomic-resolution imaging, we determine the location and identity of every atom at a grain boundary and find that different grains stitch together predominantly through pentagon-heptagon pairs. Rather than individually imaging the several billion atoms in each grain, we use diffraction-filtered imaging to rapidly map the location, orientation and shape of several hundred grains and boundaries, where only a handful have been previously reported. The resulting images reveal an unexpectedly small and intricate patchwork of grains connected by tilt boundaries. By correlating grain imaging with scanning probe and transport measurements, we show that these grain boundaries severely weaken the mechanical strength of graphene membranes but do not as drastically alter their electrical properties. These techniques open a new window for studies on the structure, properties and control of grains and grain boundaries in graphene and other two-dimensional materials.     

An integrated semiconductor device enabling non-optical genome sequencing

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.     

SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex

Cpdm (chronic proliferative dermatitis) mice develop chronic dermatitis and an immunodeficiency with increased serum IgM, symptoms that resemble those of patients with X-linked hyper-IgM syndrome and hypohydrotic ectodermal dysplasia (XHM-ED), which is caused by mutations in NEMO (NF-κB essential modulator; also known as IKBKG). Spontaneous null mutations in the Sharpin (SHANK-associated RH domain interacting protein in postsynaptic density) gene are responsible for the cpdm phenotype in mice. SHARPIN shows significant similarity to HOIL-1L (also known as RBCK1), a component of linear ubiquitin chain assembly complex (LUBAC), which induces NF-κB activation through conjugation of linear polyubiquitin chains to NEMO. Here, we identify SHARPIN as an additional component of LUBAC. SHARPIN-containing complexes can linearly ubiquitinate NEMO and activated NF-κB. Thus, we re-define LUBAC as a complex containing SHARPIN, HOIL-1L, and HOIP (also known as RNF31). Deletion of SHARPIN drastically reduced the amount of LUBAC, which resulted in attenuated TNF-α- and CD40-mediated activation of NF-κB in mouse embryonic fibroblasts (MEFs) or B cells from cpdm mice. Considering the pleomorphic phenotype of cpdm mice, these results confirm the predicted role of LUBAC-mediated linear polyubiquitination in NF-κB activation induced by various stimuli, and strongly suggest the involvement of LUBAC-induced NF-κB activation in various disorders.     

High-frequency, scaled graphene transistors on diamond-like carbon

Owing to its high carrier mobility and saturation velocity, graphene has attracted enormous attention in recent years. In particular, high-performance graphene transistors for radio-frequency (r.f.) applications are of great interest. Synthesis of large-scale graphene sheets of high quality and at low cost has been demonstrated using chemical vapour deposition (CVD) methods. However, very few studies have been performed on the scaling behaviour of transistors made from CVD graphene for r.f. applications, which hold great potential for commercialization. Here we report the systematic study of top-gated CVD-graphene r.f. transistors with gate lengths scaled down to 40 nm, the shortest gate length demonstrated on graphene r.f. devices. The CVD graphene was grown on copper film and transferred to a wafer of diamond-like carbon. Cut-off frequencies as high as 155 GHz have been obtained for the 40-nm transistors, and the cut-off frequency was found to scale as 1/(gate length). Furthermore, we studied graphene r.f. transistors at cryogenic temperatures. Unlike conventional semiconductor devices where low-temperature performance is hampered by carrier freeze-out effects, the r.f. performance of our graphene devices exhibits little temperature dependence down to 4.3 K, providing a much larger operation window than is available for conventional devices.     

Crystal structure of the β2 adrenergic receptor-Gs protein complex

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 ? outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.     

Molecular regulation of sexual preference revealed by genetic studies of 5-HT in the brains of male mice

Although the question of to whom a male directs his mating attempts is a critical one in social interactions, little is known about the molecular and cellular mechanisms controlling mammalian sexual preference. Here we report that the neurotransmitter 5-hydroxytryptamine (5-HT) is required for male sexual preference. Wild-type male mice preferred females over males, but males lacking central serotonergic neurons lost sexual preference although they were not generally defective in olfaction or in pheromone sensing. A role for 5-HT was demonstrated by the phenotype of mice lacking tryptophan hydroxylase 2 (Tph2), which is required for the first step of 5-HT synthesis in the brain. Thirty-five minutes after the injection of the intermediate 5-hydroxytryptophan (5-HTP), which circumvented Tph2 to restore 5-HT to the wild-type level, adult Tph2 knockout mice also preferred females over males. These results indicate that 5-HT and serotonergic neurons in the adult brain regulate mammalian sexual preference.     

Molecular organization of vomeronasal chemoreception

The vomeronasal organ (VNO) has a key role in mediating the social and defensive responses of many terrestrial vertebrates to species- and sex-specific chemosignals. More than 250 putative pheromone receptors have been identified in the mouse VNO, but the nature of the signals detected by individual VNO receptors has not yet been elucidated. To gain insight into the molecular logic of VNO detection leading to mating, aggression or defensive responses, we sought to uncover the response profiles of individual vomeronasal receptors to a wide range of animal cues. Here we describe the repertoire of behaviourally and physiologically relevant stimuli detected by a large number of individual vomeronasal receptors in mice, and define a global map of vomeronasal signal detection. We demonstrate that the two classes (V1R and V2R) of vomeronasal receptors use fundamentally different strategies to encode chemosensory information, and that distinct receptor subfamilies have evolved towards the specific recognition of certain animal groups or chemical structures. The association of large subsets of vomeronasal receptors with cognate, ethologically and physiologically relevant stimuli establishes the molecular foundation of vomeronasal information coding, and opens new avenues for further investigating the neural mechanisms underlying behaviour specificity.