In vivo DNA methylation editing in zebrafish.

The CRISPR/dCas9-based epigenome editing technique has driven much attention. Fused with a catalytic domain from Dnmt or Tet protein, the CRISPR/dCas9-DnmtCD or -TetCD systems possess the targeted DNA methylation editing ability and have established a series of in vitro and in vivo disease models. However, no publication has been reported on zebrafish (Danio rerio), an important animal model in biomedicine. The present study demonstrated that CRISPR/dCas9-Dnmt7 and -Tet2 catalytic domain fusions could site-specifically edit genomic DNA methylation in vivo in zebrafish and may serve as an efficient toolkit for DNA methylation editing in the zebrafish model.

Toxicological damages on copper exposure to IgM+ B cells of Nile tilapia (Oreochromis niloticus) and mitigation of its adverse effects by ß-glucan administration.

Present investigation was carried out to study toxicological damages of copper exposure and mitigation of its adverse effects with ß-glucan administration in IgM+ B cells which processes multiple roles similar to macrophages in Nile tilapia (Oreochromis niloticus). IgM+ B cells were pretreated with ß-glucan (25 µg/mL) for 24 h before exposed to cupric oxide nanoparticles (CuO NPs) or cupric chloride (Cu ions) at the doses of 0, 5, 10, and 20 µg/mL for 24 h, respectively. Our results demonstrated that ß-glucan increased reduced glutathione (GSH) to against oxidative damage from CuO NPs and Cu ions exposure in IgM+ B cells. The apoptosis process through mitochondrial signaling pathway was depressed in IgM+ B cells since the mitochondrial membrane potential (ΔΨm) was protected from copper exposure by ß-glucan treatment. Furthermore, the inhibition on phagocytic abilities of IgM+ B cells caused by copper exposure could be enhanced with ß-glucan treatment via evaluation of microspheres and bioparticles uptake and LPS-induced NO production. Importantly, ß-glucan might participate in immunomodulation in IgM+ B cells through B cell antigen receptor (BCR) to suppress toxicological effect derived from copper exposure. Taken together, this study provides more information on the toxicological damages in IgM+ B cells upon copper exposure and explains the molecular mechanism to reverse adverse effects caused by copper exposure with ß-glucan administration.

ß-glucan mitigation on toxicological effects in monocytes/macrophages of Nile tilapia (Oreochromis niloticus) following copper exposure.

The protective effect of ß-glucan against toxicological effects caused by copper oxide nanoparticles (Cu NPs) and copper ions (Cu ions) were studied in monocytes/macrophages (MO/MФ) of Nile tilapia (Oreochromis niloticus). Our results demonstrated that CuO NPs and Cu ions exposure aroused strong oxidative lesion in MO/MФ by detection of cellular reactive oxygen species (ROS) and reduced glutathione (GSH), as well as identification of several antioxidant-related cytokines. Meanwhile, the serious pro-inflammatory responses were accompanied during the processes of oxidative lesion by TNFα, IL-1ß, and IL-6 genes validation. Copper induced MO/MФ underwent apoptosis through mitochondrial signaling pathway by mitochondrial membrane potential (ΔΨm) detection and Bax, Bcl-2, Cyt-c, Apaf-1, Caspase 9, Caspase 3 genes validation. Furthermore, the phagocytic abilities were inhibition in MO/MФ by evaluation of microspheres (0.5 and 1.0 µm beads) and bioparticles (S. agalactiae and A. hydrophila) uptake and LPS-induced NO production. However, ß-glucan might participate in immunomodulation through C-type lectin receptor (CLR) and complement receptor 3 (CR3) to suppress pro-inflammatory responses, thereby revered all the copper induced aforementioned adverse effects in MO/MΦ. Taken together, our results provide insights on the mechanisms through ß-glucan administration to mitigate toxicological effects of CuO NPs and Cu ions exposure to the MO/MΦ, which will benefit aspects related to fish farming and aquaculture production.

Vitamin C suppresses toxicological effects in MO/MФ and IgM+ B cells of Nile tilapia (Oreochromis niloticus) upon copper exposure.

Copper (Cu), as an essential micronutrient in human and animal metabolism, easily spreads and excessively accumulates in rearing water, which make it more susceptible to fish farms and threatens the health of aquatic animals. In this issue, the protective effect of vitamin C against oxidative damage caused by copper exposure was studied in monocytes/macrophages (MO/MФ) and IgM+ B cells of Nile tilapia (Oreochromis niloticus), the cell types possessing phagocytic activities. The significant increase of ROS level and up-regulation of proinflammatory factors accompanied by depletion of GSH and down-regulation of antioxidative molecules in MO/MФ and IgM+ B cells, when stressed with CuO NPs or Cu ions, indicated the induction of oxidative damage due to the toxicological effects with copper exposure. Copper induced cell apoptosis through mitochondrial-dependent pathway in these two cell populations was demonstrated with disruption of mitochondrial membrane potential (ΔΨm) and activation of apoptosis factor. Furthermore, the phagocytic abilities for microspheres and bioparticle uptake significantly decreased in these two cell populations upon CuO NPs or Cu ions; meanwhile, antigen presentation of MO/MФ and antibody production of IgM+ B cells were also inhibited. However, vitamin C supplementation reversed all these biochemical indices, as well as cell apoptosis and phagocytic abilities in MO/MФ and IgM+ B cells that were induced by CuO NPs or Cu ions. In conclusion, these results revealed that vitamin C exerts cytoprotective effects against oxidative damage through its antioxidant properties and may be of therapeutic use in preventing toxicological effects caused by copper exposure.

Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM.

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5'-NNG-3' PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.