Summary The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, sgRNA parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for the first time a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair, and achieved an overall mutagenesis rate significantly higher than previously reported. Our work presents the most comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

The Taiwan Biobank (TWB) aims to build a nationwide research database that integrates genomic/epigenomic profiles, lifestyle patterns, dietary habits, environmental exposure history and long-term health outcomes of 300,000 residents of Taiwan. We describe here an investigation of the population structure of Han Chinese on this Pacific island using genotype data of 591,048 SNPs in an initial freeze of 10,801 unrelated TWB participants. In addition to the North-South cline reported in other Han Chinese populations, we find the Taiwanese Han Chinese clustered into three cline groups: 5% were of northern Han Chinese ancestry, 79.9% were of southern Han Chinese ancestry, and 14.5% belonged to a third (T) group. We also find that this T group is genetically distinct from neighbouring Southeast Asians and Austronesian tribes but similar to other southern Han Chinese. Interestingly, high degree of LD between HLA haplotype A*33:03-B*58:01, an MHC allele being of pathological relevance, and SNPs across the MHC region was observed in subjects with T origin, but not in other Han Chinese. This suggested the T group individuals may have experienced evolutionary events independent from the other southern Han Chinese. Based on the newly-discovered population structure, we detect different loci susceptible to type II diabetes in individuals with southern and northern Han Chinese ancestries. Finally, as one of the largest dataset currently available for the Chinese population, genome-wide statistics for the 10,810 subjects are made publicly accessible through Taiwan View (https://taiwanview.twbiobank.org.tw/index; date last accessed October 14, 2016) to encourage future genetic research and collaborations with the island Taiwan.

Tumor necrosis factor α (TNFα) is a pleiotropic cytokine which signals through TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Emerging evidence has demonstrated that TNFR1 is ubiquitously expressed on almost all cells, while TNFR2 exhibits a limited expression, predominantly on regulatory T cells (Tregs). In addition, the signaling pathway by sTNF via TNFR1 mainly triggers pro-inflammatory pathways, and mTNF binding to TNFR2 usually initiates immune modulation and tissue regeneration. TNFα plays a critical role in upregulation or downregulation of Treg activity. Deficiency in TNFR2 signaling is significant in various autoimmune diseases. An ideal therapeutic strategy for autoimmune diseases would be to selectively block the sTNF/TNFR1 signal through the administration of sTNF inhibitors, or using TNFR1 antagonists while keeping the TNFR2 signaling pathway intact. Another promising strategy would be to rely on TNFR2 agonists which could drive the expansion of Tregs and promote tissue regeneration. Design of these therapeutic strategies targeting the TNFR1 or TNFR2 signaling pathways holds promise for the treatment of diverse inflammatory and degenerative diseases.

Tumor Necrosis Factor (TNF) α is a multifunctional cytokine with pro-inflammatory and anti-inflammatory characteristics. Increasing evidence suggests that thymus-derived, natural regulatory T cells (nTreg) express a remarkably high level of TNF Receptor 2 (TNFR2) and TNFα modulates the number or function of nTreg via TNFR2 in autoimmune diseases. Nonetheless, Treg cells consist of at least nTreg and iTreg that are induced in the periphery or in vitro and two subsets may have different biological characteristics. However, the role of TNF-TNFR signaling in development and function of these iTreg cells is less clear. In this study, we systemically studied the effect of TNFα and its receptor signals on iTreg differentiation, proliferation, and function in vitro and in vivo. We further investigated the expression and requirement of TNFR1 or TNFR2 expression on iTreg by utilizing TNFR1−/− and TNFR2−/− mice. We found that exogenous TNFα facilitated iTreg differentiation and function in vitro. TNFR2 deficiency hampered iTreg differentiation, proliferation, and function, while TNFR1 deficiency decreased the differentiation of inflammatory T cells such as Th1 and Th17 cells but maintained the regulatory capabilities of iTreg both in vitro and in vivo. Using colitis model, we also revealed TNFR2 but not TNFR1 deficiency compromised the iTreg functionality. Interestingly, inflammation affects TNFR expression on nTreg but not iTreg subset. Our results demonstrate that exogenous TNFα may enhance the differentiation and function of iTreg via TNFR2 signaling. The expression of TNFR2 on Treg might be downregulated in some autoimmune diseases, accompanied by an increased level of TNFR1. Thus, TNFR2 agonists or TNFR1-specific antagonists hold a potential promise for clinical application in treating patients with autoimmune diseases.