Although Cas9 nucleases are remarkably diverse in microorganisms, the range of genomic sequences targetable by a CRISPR/Cas9 system is restricted by the requirement of a short protospacer adjacent motif (PAM) at the target site. Here, we generate a group of chimeric Cas9 (cCas9) variants by replacing the key region in the PAM interaction (PI) domain of Staphylococcus aureus Cas9 (SaCas9) with the corresponding region in a panel of SaCas9 orthologs. By using a functional assay at target sites with different nucleotide recombinations at PAM position 3–6, we identify several cCas9 variants with expanded recognition capability at NNVRRN, NNVACT, NNVATG, NNVATT, NNVGCT, NNVGTG, and NNVGTT PAM sequences. In summary, we provide a panel of cCas9 variants accessible up to 1/4 of all the possible genomic targets in mammalian cells.

BACKGROUND Hydrocolloids are the most commonly used additive in the processing of surimi products. However, the effect of hydrocolloids on surimi protein conformation has not been reported, and the level of hydrocolloids may be a key factor influencing the quality of surimi. Therefore, this study investigated the effect of curdlan, xanthan gum, κ‐carrageenan, and gelatin at various levels on gel properties and protein conformation of surimi from silver carp. RESULTS Addition of curdlan, κ‐carrageenan, or gelatin at lower level could significantly promote gel strength, textural profiles, and water holding capacity (WHC) of the surimi gels. However, gel strength and WHC gradually decreased with increasing amount of xanthan gum added. The addition of curdlan or κ‐carrageenan remarkably increased the whiteness of surimi gel, but the whiteness decreased when the concentration of κ‐carrageenan reached 5 g kg−1. Along with the increase of curdlan, κ‐carrageenan, or gelatin concentration, the index of hydrophobic interaction and hydrogen bonds first increased and then decreased, whereas index of ionic bonds first decreased and then increased. According to Raman spectroscopy data, a small content of curdlan or κ‐carrageenan promoted the conformational transition of surimi protein from α‐helix to β‐sheet, leading to the changes in gel properties of surimi gels. Scanning electron microscopy photographs showed surimi gels added with 4 g kg−1 curdlan or 2 g kg−1 κ‐carrageenan had a finer and denser network structure. CONCLUSION Curdlan or κ‐carrageenan at an appropriate concentration is a potential modifier to effectively improve the quality of surimi products. © 2020 Society of Chemical Industry

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Background Our group previously found that the transmembrane protein 232 (TMEM232) gene was associated with atopic dermatitis (AD) by Genome-wide association study and fine mapping study. However, its function is unclear so far. Objective To investigate the roles and mechanisms of TMEM232 in AD. Methods The expression of TMEM232 was investigated in skin lesions of AD patients, MC903-induced AD mouse model, human primary keratinocytes and HaCaT cells stimulated with different inflammatory factors. The role of TMEM232 in AD was analyzed in HaCaT cells and Tmem232 knockout (Tmem232-/-) mice. Tmem232-specific small interfering RNA (siRNA) was used to evaluate its therapeutic potential in the AD mouse model. Results The expression of TMEM232 was significantly increased in skin lesions of AD patients，MC903-induced AD mouse model and human primary keratinocytes and HaCaT cells stimulated with different inflammatory factors compared to controls. In the presence of MC903, Tmem232-/- mice exhibited significantly reduced dermatitis severity, mast cell infiltration in the back, and expression of Th1 and Th2-related inflammatory factors in skin tissue compared with WT mice. In vitro and in vivo experiments further showed that the upregulation of TMEM232 in AD exacerbated inflammation response through activating the pathway of nuclear factor-κB and signal transducer and activator of transcription (STAT) 3, and was regulated by IL4/STAT6 axis, which formed a self-amplifying loop. Finally, topical application of Tmem232-siRNA markedly ameliorated AD-like lesions in the AD model. Conclusion The study first outlines the function of TMEM232. It is involved in regulating inflammation in AD and may be a potential target for AD treatment.