Foreign DNA capture during CRISPR–Cas adaptive immunity

Nature 527, 7579 (2015). doi:10.1038/nature15760

Authors: James K. Nuñez, Lucas B. Harrington, Philip J. Kranzusch, Alan N. Engelman & Jennifer A. Doudna

Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1–Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

Quantum physics: Death by experiment for local realism

Nature 526, 7575 (2015). doi:10.1038/nature15631

Authors: Howard Wiseman

A fundamental scientific assumption called local realism conflicts with certain predictions of quantum mechanics. Those predictions have now been verified, with none of the loopholes that have compromised earlier tests. See Letter p.682

Loophole-free Bell inequality violation using electron spins separated by 1.3 kilometres

Nature 526, 7575 (2015). doi:10.1038/nature15759

Authors: B. Hensen, H. Bernien, A. E. Dréau, A. Reiserer, N. Kalb, M. S. Blok, J. Ruitenberg, R. F. L. Vermeulen, R. N. Schouten, C. Abellán, W. Amaya, V. Pruneri, M. W. Mitchell, M. Markham, D. J. Twitchen, D. Elkouss, S. Wehner, T. H. Taminiau & R. Hanson

More than 50 years ago, John Bell proved that no theory of nature that obeys locality and realism can reproduce all the predictions of quantum theory: in any local-realist theory, the correlations between outcomes of measurements on distant particles satisfy an inequality that can be violated if the particles are entangled. Numerous Bell inequality tests have been reported; however, all experiments reported so far required additional assumptions to obtain a contradiction with local realism, resulting in ‘loopholes’. Here we report a Bell experiment that is free of any such additional assumption and thus directly tests the principles underlying Bell’s inequality. We use an event-ready scheme that enables the generation of robust entanglement between distant electron spins (estimated state fidelity of 0.92 ± 0.03). Efficient spin read-out avoids the fair-sampling assumption (detection loophole), while the use of fast random-basis selection and spin read-out combined with a spatial separation of 1.3 kilometres ensure the required locality conditions. We performed 245 trials that tested the CHSH–Bell inequalityS ≤ 2 and found S = 2.42 ± 0.20 (where S quantifies the correlation between measurement outcomes). A null-hypothesis test yields a probability of at most P = 0.039 that a local-realist model for space-like separated sites could produce data with a violation at least as large as we observe, even when allowing for memory in the devices. Our data hence imply statistically significant rejection of the local-realist null hypothesis. This conclusion may be further consolidated in future experiments; for instance, reaching a value of P = 0.001 would require approximately 700 trials for an observed S = 2.4. With improvements, our experiment could be used for testing less-conventional theories, and for implementing device-independent quantum-secure communication and randomness certification.

Thalamic control of sensory selection in divided attention

Nature 526, 7575 (2015). doi:10.1038/nature15398

Authors: Ralf D. Wimmer, L. Ian Schmitt, Thomas J. Davidson, Miho Nakajima, Karl Deisseroth & Michael M. Halassa

How the brain selects appropriate sensory inputs and suppresses distractors is unknown. Given the well-established role of the prefrontal cortex (PFC) in executive function, its interactions with sensory cortical areas during attention have been hypothesized to control sensory selection. To test this idea and, more generally, dissect the circuits underlying sensory selection, we developed a cross-modal divided-attention task in mice that allowed genetic access to this cognitive process. By optogenetically perturbing PFC function in a temporally precise window, the ability of mice to select appropriately between conflicting visual and auditory stimuli was diminished. Equivalent sensory thalamocortical manipulations showed that behaviour was causally dependent on PFC interactions with the sensory thalamus, not sensory cortex. Consistent with this notion, we found neurons of the visual thalamic reticular nucleus (visTRN) to exhibit PFC-dependent changes in firing rate predictive of the modality selected. visTRN activity was causal to performance as confirmed by bidirectional optogenetic manipulations of this subnetwork. Using a combination of electrophysiology and intracellular chloride photometry, we demonstrated that visTRN dynamically controls visual thalamic gain through feedforward inhibition. Our experiments introduce a new subcortical model of sensory selection, in which the PFC biases thalamic reticular subnetworks to control thalamic sensory gain, selecting appropriate inputs for further processing.

CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase

Nature 526, 7575 (2015). doi:10.1038/nature15510

Authors: Weiwei He, Ge Bai, Huihao Zhou, Na Wei, Nicholas M. White, Janelle Lauer, Huaqing Liu, Yi Shi, Calin Dan Dumitru, Karen Lettieri, Veronica Shubayev, Albena Jordanova, Velina Guergueltcheva, Patrick R. Griffin, Robert W. Burgess, Samuel L. Pfaff & Xiang-Lei Yang

Selective neuronal loss is a hallmark of neurodegenerative diseases, which, counterintuitively, are often caused by mutations in widely expressed genes. Charcot–Marie–Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies. A subtype of these diseases—CMT type 2D (CMT2D)—is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-transfer RNA (tRNA) synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons, leading to deficits in distal motor function. How mutations in GlyRS (GlyRSCMT2D) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRSCMT2D acquires a neomorphic binding activity that directly antagonizes an essential signalling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRSCMT2D to bind the neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRSCMT2D that antagonizes the VEGF–Nrp1 interaction, and indicate that the VEGF–Nrp1 signalling axis is an actionable target for treating CMT2D.

Yap-dependent reprogramming of Lgr5+ stem cells drives intestinal regeneration and cancer

Nature 526, 7575 (2015). doi:10.1038/nature15382

Authors: Alex Gregorieff, Yu Liu, Mohammad R. Inanlou, Yuliya Khomchuk & Jeffrey L. Wrana

The gut epithelium has remarkable self-renewal capacity that under homeostatic conditions is driven by Wnt signalling in Lgr5+ intestinal stem cells (ISCs). However, the mechanisms underlying ISC regeneration after injury remain poorly understood. The Hippo signalling pathway mediates tissue growth and is important for regeneration. Here we demonstrate in mice that Yap, a downstream transcriptional effector of Hippo, is critical for recovery of intestinal epithelium after exposure to ionizing radiation. Yap transiently reprograms Lgr5+ ISCs by suppressing Wnt signalling and excessive Paneth cell differentiation, while promoting cell survival and inducing a regenerative program that includes Egf pathway activation. Accordingly, growth of Yap-deficient organoids is rescued by the Egfr ligand epiregulin, and we find that non-cell-autonomous production of stromal epiregulin may compensate for Yap loss in vivo. Consistent with key roles for regenerative signalling in tumorigenesis, we further demonstrate that Yap inactivation abolishes adenomas in the ApcMin mouse model of colon cancer, and that Yap-driven expansion of Apc−/− organoids requires the Egfr module of the Yap regenerative program. Finally, we show that in vivo Yap is required for progression of early Apc mutant tumour-initiating cells, suppresses their differentiation into Paneth cells, and induces a regenerative program and Egfr signalling. Our studies reveal that upon tissue injury, Yap reprograms Lgr5+ ISCs by inhibiting the Wnt homeostatic program, while inducing a regenerative program that includes activation of Egfr signalling. Moreover, our findings reveal a key role for the Yap regenerative pathway in driving cancer initiation.

Abstract thoughts

Nature 526, 7574 (2015). doi:10.1038/526475b

Scientists, meeting organizers and the media must take care with preliminary findings.

Pick and mix

Nature 526, 7574 (2015). doi:10.1038/526756a

Food regulators are right to place new forms of data on the safety menu.

Planetary science: Pluto hosts wildly varying terrain

Nature 526, 7574 (2015). doi:10.1038/526478a

The first published findings from NASA's New Horizons mission to Pluto confirm that the dwarf planet has geological features that resemble those found on Mars and various moons in the Solar System.NASA's spacecraft flew past Pluto in July, sending back reams of data that