No abstract

In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits.

Daily 24-hour PM 2.5 samples were collected continuously from January 1 to December 31, 2010. Elemental concentrations from Al to Pb were obtained using particle induced X-ray emission (PIXE) method. This was the first full year continuous daily PM 2.5 elemental composition dataset in Beijing. Source apportionment analysis was conducted on this dataset using the positive matrix factorization method. Seven sources and their contributions to the total PM 2.5 mass were identified and quantified. These include secondary sulphur-13.8 μg/m 3 ) compared to those in the spring and summer (9.6 and 8.0 μg/m 3 , respectively). Secondary sulphur contributed the most in the summer while vehicle exhaust and metal processing sources did not show any clear seasonal pattern. The different seasonal highs and lows from different sources compensated each other. This explains the very small seasonal variations (< 20%) in the total PM 2.5 .