The DNA mutation produced by cellular repair of a CRISPR/Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, and depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR/Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions, or longer microhomology-mediated deletions. Each gRNA has an individual cell-line dependent bias toward particular outcomes. We uncover sequence determinants of the produced mutations, and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

Brain-derived neurotrophic factor (BDNF) and other neurotrophins have a vital role in the development of the rat and mouse nervous system by influencing the expression of many specific genes that promote differentiation, cell survival, synapse formation and, later, synaptic plasticity. Although nitric oxide (NO) is known to be an important mediator of BDNF signalling in neurons, the mechanisms by which neurotrophins influence gene expression during development and plasticity remain largely unknown. Here we show that BDNF triggers NO synthesis and S-nitrosylation of histone deacetylase 2 (HDAC2) in neurons, resulting in changes to histone modifications and gene activation. S-nitrosylation of HDAC2 occurs at Cys 262 and Cys 274 and does not affect deacetylase activity. In contrast, nitrosylation of HDAC2 induces its release from chromatin, which increases acetylation of histones surrounding neurotrophin-dependent gene promoters and promotes transcription. Notably, nitrosylation of HDAC2 in embryonic cortical neurons regulates dendritic growth and branching, possibly by the activation of CREB (cyclic-AMP-responsive-element-binding protein)-dependent genes. Thus, by stimulating NO production and S-nitrosylation of HDAC2, neurotrophic factors promote chromatin remodelling and the activation of genes that are associated with neuronal development.

Almost 25 years after the first report that glutamate can activate receptors coupled to heterotrimeric G-proteins, tremendous progress has been made in the field of metabotropic glutamate receptors. Now, eight members of this family of glutamate receptors, encoded by eight different genes that share distinctive structural features have been identified. The first cloned receptor, the metabotropic glutamate ( mGlu) receptor mGlu1 has probably been the most extensively studied mGlu receptor, and in many respects it represents a prototypical subtype for this family of receptors. Its biochemical, anatomical, physiological, and pharmacological characteristics have been intensely investigated. Together with subtype 5, mGlu1 receptors constitute a subgroup of receptors that couple to phospholipase C and mobilize Ca(2+) from intracellular stores. Several alternatively spliced variants of mGlu1 receptors, which differ primarily in the length of their C-terminal domain and anatomical localization, have been reported. Use of a number of genetic approaches and the recent development of selective antagonists have provided a means for clarifying the role played by this receptor in a number of neuronal systems. In this article we discuss recent advancements in the pharmacology and concepts about the intracellular transduction and pathophysiological role of mGlu1 receptors and review earlier data in view of these novel findings. The impact that this new and better understanding of the specific role of these receptors may have on novel treatment strategies for a variety of neurological and psychiatric disorders is considered

We have recently shown that IL-10 fails to trigger Stat3 and Stat1 tyrosine phosphorylation in freshly isolated human neutrophils. In this study, we report that IL-10 can nonetheless induce Stat3 tyrosine phosphorylation and the binding of Stat1 and Stat3 to the IFN-γ response region or the high-affinity synthetic derivative of the c-sis-inducible element in neutrophils that have been cultured for at least 3 h with LPS. Similarly, the ability of IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was dramatically enhanced in cultured neutrophils and, as a result, translated into the SOCS-3 protein. Since neutrophils’ acquisition of responsiveness to IL-10 required de novo protein synthesis, we assessed whether expression of IL-10R1 or IL-10R2 was modulated in cultured neutrophils. We detected constitutive IL-10R1 mRNA and protein expression in circulating neutrophils, at levels which were much lower than those observed in autologous monocytes or lymphocytes. In contrast, IL-10R2 expression was comparable in both cell types. However, IL-10R1 (but not IL-10R2) mRNA and protein expression was substantially increased in neutrophils stimulated by LPS. The ability of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3 synthesis and to regulate IL-1 receptor antagonist and macrophage-inflammatory protein 1β release in LPS-treated neutrophils correlated with this increased IL-10R1 expression, and was abolished by neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results demonstrate that the capacity of neutrophils to respond to IL-10, as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and modulation of cytokine production, is very dependent on the level of expression of IL-10R1.