Increased anxiety was found in serpini1 knockout zebrafish larval

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.

One of the most commonly used behavioral endpoints measured in preclinical studies using rodent models is thigmotaxis (or "wall-hugging"). Thigmotaxis is a well-validated index of anxiety in animals and humans. While assays measuring thigmotaxis in adult zebrafish have been developed, a thigmotaxis assay has not yet been validated in larval zebrafish. Here we present a novel assay for measurement of thigmotaxis in zebrafish larvae that is triggered by a sudden change in illumination (i.e. sudden light-to-darkness transition) and performed in a standard 24-well plate. We show that zebrafish larvae as young as 5 days post fertilization respond to this challenge by engaging in thigmotaxis. Thigmotaxis was significantly attenuated by anxiolytic (diazepam) and significantly enhanced by anxiogenic (caffeine) drugs, thus representing the first validated thigmotaxis assay for larval zebrafish. We also show that exposure to sudden darkness per se may represent an anxiogenic situation for larval zebrafish since less contrasting light-to-darkness transitions (achieved by lowering darkness degrees) significantly decreased thigmotaxis levels in a manner similar to what was achieved with diazepam. These findings suggest that stimuli such as exposure to sudden darkness could be used proficiently to trigger the expression of anxiety-like behaviors in laboratory settings. In sum, this is a versatile protocol allowing testing of both anxiolytic and anxiogenic drugs in a cost-effective manner (only 10 min). This assay is also amenable to medium to high-throughput capacity while constituting a valuable tool for stress and central nervous system research as well as for preclinical drug screening and discovery. Copyright © 2011 Elsevier B.V. All rights reserved.

Here we present a strategy to determine the genetic basis of variance in complex phenotypes that arise from natural, as opposed to induced, genetic variation in mice. We show that a commercially available strain of outbred mice, MF1, can be treated as an ultrafine mosaic of standard inbred strains and accordingly used to dissect a known quantitative trait locus influencing anxiety. We also show that this locus can be subdivided into three regions, one of which contains Rgs2, which encodes a regulator of G protein signaling. We then use quantitative complementation to show that Rgs2 is a quantitative trait gene. This combined genetic and functional approach should be applicable to the analysis of any quantitative trait.