Project description:To generate a test data to facilitate development of toolkit for off-target nomination data analysis, a library consisting >81K in silico predicted on- and off-target regions for an EMX1 gRNA are synthesized and cleaved in vitro with SpCas9. The cleaved products are used to build NGS library followed by sequencing leading to >13 million reads. About 99% of examined regions are covered by 10 or more valid UMIs. More than 90% of valid UMIs from on-target region support cleavage. About 500 off-target regions have a score greater than 0.1, including 7 off-target regions with score exceeding one.
Project description:Altered DNA methylation is a crucial epigenetic event in hepatocellular carcinoma (HCC) development and progression. Through methylation-transcriptomic analysis, we identified a set of sixty potential DNA methylation-based epidriver genes. This set of genes focuses on the hypermethylation of EMX1, which is frequently observed in hepatobiliary tumors. Despite of its frequent occurrence, the function of EMX1 remains largely unknown. By utilizing bisulfite-next-generation sequencing, we have detected EMX1 DNA hypermethylation on the gene body, which is positively correlated with EMX1 mRNA expression. Further analysis revealed that EMX1 mRNA terminal exon splicing in HCC generated two protein isoforms: EMX1 full length (EMX1-FL) and alternative terminal exon splicing isoform (EMX1-X1). Cellular functional assays demonstrated that gain-of-function EMX1-FL, but not EMX1-X1, induced HCC cells migration and invasion while silencing EMX1-FL inhibited HCC cells motility. This result was further validated by in vivo tumor metastasis models. Mechanistically, EMX1-FL bound to EGFR promoter, promoting EGFR transcription and activating EGFR-ERK signaling to trigger tumor metastasis. Therefore, EGFR may be a potential therapeutic target for EMX1-high expression HCC. Our work illuminated the crucial role of gene body hypermethylation-activated EMX1-FL in promoting tumorigenesis and metastasis in HCC. These findings pave the way for targeting the EMX1-EGFR axis in HCC tumorigenicity and metastasis.
Project description:This is RNA sequencing performed with embryonic day 12 (E12) microdissected mouse cortices with the following genotypes: Emx1 Cre/ +; Zbtb7a +/+ (WT), Emx1 Cre/+; Zbtb7a fl/+ (cHet) and Emx1 Cre/+; Zbtb7a fl/fl (cKO). The goal of this study was to understand the impact ot Zbtb7a knockout during cortical development.
Project description:the goal of the study was to compare gene expression between control and Pbx1; Emx1-cre mutant corteces at e15.5 E15.5 whole cortex was dissected for the analysis. Control embryo genotype was Pbx1F/+. Mutant embryo genotype was Pbx1F/-; Emx1-cre. 4 corteces of each genotype were used for the analysis: 4 controls and 4 mutants, 8 samples total.
Project description:the goal of the study was to compare gene expression between control and Pbx1; Emx1-cre mutant corteces at e12.5 E12.5 whole cortex was dissected for the analysis. Control embryo genotype was Pbx1F/+. Mutant embryo genotype was Pbx1F/-; Emx1-cre. 4 corteces of each genotype were used for the analysis: 4 controls and 4 mutants, 8 samples total.
Project description:This is RNA sequencing performed with embryonic day 12 (E12) microdissected mouse cortices with the following genotypes: Emx1 Cre/ +; Zbtb7a +/+ (WT) and Emx1 Cre/+; Zbtb7a fl/fl (cKO). The goal of this study was to understand the impact ot Zbtb7a knockout during cortical development.