In order to elucidate the functional organization of the genome, it is vital to directly visualize the interactions between genomic elements in living cells. For this purpose, we engineered the Cas9 protein from Staphylococcus aureus (SaCas9) for the imaging of endogenous genomic loci, which showed a similar robustness and efficiency as previously reported for Streptococcus pyogenes Cas9 (SpCas9). Imaging readouts allowed us to characterize the DNA-binding activity of SaCas9 and to optimize its sgRNA scaffold. Combining SaCas9 and SpCas9, we demonstrated two-color CRISPR imaging with the capability to resolve genomic loci spaced by <300 kb. Combinatorial color-mixing further enabled us to code multiple genomic elements in the same cell. Our results highlight the potential of combining SpCas9 and SaCas9 for multiplexed CRISPR-Cas9 applications, such as imaging and genome engineering.

Highlights d The dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to Nurr1 d DHI forms a covalent adduct with Nurr1, reacting as the indolequinone with Cys566 d The Nurr1-metabolite structure reveals a previously unreported ligand-binding pocket d DHI stimulates the transcription of Nurr1 target genes underlying dopamine homeostasis

BackgroundLarval zebrafish, with a simple and transparent vertebrate brain composed of ~100 K neurons, is well suited for deciphering entire neural circuit activity underlying behavior. Moreover, their small body size (~4–5 mm in length) is compatible with 96-well plates, making larval zebrafish amenable to high content screening. Despite these attractive features, there is a scarcity of behavioral characterizations in larval zebrafish compared to other model organisms as well as adult zebrafish.ResultsIn this study, we have characterized the physiological and behavioral responses of larval zebrafish to several easily amenable stimuli, including heat, cold, UV, mechanical disturbance (MD), and social isolation (SI). These stimuli are selected based on their perceived aversive nature to larval zebrafish. Using a light/dark choice paradigm, in which larval zebrafish display an innate dark avoidance behavior (i.e. scotophobia), we find that heat, cold and UV stimuli significantly enhance their dark avoidance with heat having the most striking effect, whereas MD and SI have little influence on the behavior. Surprisingly, using the cortisol assay, a physiological measure of stress, we uncover that all stimuli but heat and SI significantly increase the whole body cortisol levels.ConclusionThese results identify a series of stressors that can be easily administered to larval zebrafish. Those stimuli that elicit differential responses at behavioral and physiological levels warrant further studies at circuit levels to understand the underlying mechanisms. The findings that various stressors enhance while anxiolytics attenuate dark avoidance further reinforce that the light/dark preference behavior in larval zebrafish is fear/anxiety-associated.

Abstract:We have developed a new open-top selective plane illumination microscope (SPIM) compatible with microfluidic devices, multi-well plates, and other sample formats used in conventional inverted microscopy. Its key element is a water prism that compensates for the aberrations introduced when imaging at 45 degrees through a coverglass. We have demonstrated its unique high-content imaging capability by recording Drosophila embryo development in environmentally-controlled microfluidic channels and imaging zebrafish embryos in 96-well plates. We have also shown the imaging of C. elegans and moving Drosophila larvae on coverslips. Hillman, "Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms," Nat. Photonics 9(2), 113-119 (2015 an open-source platform for biological-image analysis," Nat. Methods 9(7), 676-682 (2012).