Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by augmented type I interferon signaling. High-throughput technologies have identified plenty of SLE susceptibility single-nucleotide polymorphisms (SNPs) yet the exact roles of most of them are still unknown. Functional studies are principally focused on SNPs in the coding regions, with limited attention paid to the SNPs in non-coding regions. Long non-coding RNAs (lncRNAs) are important players in shaping the immune response and show relationship to autoimmune diseases. In order to reveal the role of SNPs located near SLE related lncRNAs, we performed a transcriptome profiling of SLE patients and identified linc00513 as a significantly over expressed lncRNA containing functional SLE susceptibility loci in the promoter region. The risk-associated G allele of rs205764 and A allele of rs547311 enhanced linc00513 promoter activity and related to increased expression of linc00513 in SLE. We also identified linc00513 to be a novel positive regulator of type I interferon pathway by promoting the phosphorylation of STAT1 and STAT2. Elevated linc00513 expression positively correlated with IFN score in SLE patients. Linc00513 expression was higher in active disease patients than those inactive ones. In conclusion, our data identify two functional promoter variants of linc00513 that contribute to increased level of linc00513 and confer susceptibility on SLE. The study provides new insights into the genetics of SLE and extends the role of lncRNAs in the pathogenesis of SLE.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.