SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution.

CRISPR mutagenesis screens conducted with SpCas9 and other nucleases have identified certain cis-regulatory elements and genetic variants but at a limited resolution due to the absence of protospacer adjacent motif (PAM) sequences. Here, leveraging the broad targeting scope of the near-PAMless SpRY variant, we have demonstrated that saturated SpRY mutagenesis and base editing screens can faithfully identify functional regulatory elements and essential genetic variants for target gene expression at single-base resolution. We further extended this methodology to investigate a genome-wide association study (GWAS) locus at 10q22.1 associated with a red blood cell trait, where we identified potential enhancers regulating HK1 gene expression, despite not all of these enhancers exhibiting typical chromatin signatures. More importantly, our saturated base editing screens pinpoint multiple causal variants within this locus that would otherwise be missed by Bayesian statistical fine-mapping. Our approach is generally applicable to functional interrogation of all non-coding genomic elements while complementing other high-coverage CRISPR screens.

Exploration on Molecular Mechanism of Reversal Effect of Compound Danshen Tablets on Hepatic Fibrosis Based on Network Pharmacology.

Objective: To research the molecular mechanism of compound Danshen tablets in the treatment of hepatic fibrosis through network pharmacology. Methods: Traditional Chinese medicine systems pharmacology (TCMSP) and online Mendelian inheritance in man (OMIM) databases were searched for compound Danshen tablets' active ingredients o and hepatic fibrosis-related genes. The network enrichment of the targets of "herb-compound-target" was visualized and analyzed using Cytoscape software. Then, the screened target genes were used to construct a protein-protein interaction network. The DAVID enrichment database (the database for annotation, visualization, and integrated discovery) was adopted for GO (Gene Ontology) enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment of vital nodes. Results: The results yielded 234 targets of compound Danshen tablets; ten important targets (TNF, IL-10, TGF-ß1, EGF, CXCL16, CCL21, SERPINB5, SERPINA1, SOD2, and PPIG) for reversing hepatic fibrosis; and four core targets (TNF, IL-10, TGF-1, and EGF). In addition, KEGG enrichment analysis showed that compound Danshen tablets mainly involved FoxO and MAPK signaling pathways, as the key signaling pathways in the treatment of hepatic fibrosis. Conclusion: TNF, IL-10, TGF-1, and EGF and FOXO and MAPK signaling pathways play a key role in the pathogenesis of hepatic fibrosis. Based on the results of this study, the mechanism of action of compound Danshen tablets in the treatment of hepatic fibrosis may be associated with the regulation of FoxO and MAPK signaling pathways and inhibition of TNF, IL-10, TGF-1, and EGF.