RNA-seq Analysis Of The SCN1A-KO Model Based On CRISPR/Cas9 Genome Editing Technology

Objective The focus of this study was to establish a SCN1A gene-stable knockout cell line in vitro using the CRISPR/Cas9 gene editing technology.RNA-seq was then used to analyze changes in gene expression profiles of SCN1 Aknockout cells.The association between SCNIA and DS onset was analyzed by detecting changes in mRNA levels and hopefully provided some clues for the treatment of Dravet syndromeMethods ? The sgRNA was designed based on the SCNIA promoter sequence to construct a CRISPR/Cas9 knockout plasmid(pX459-SCN1A).? The pX459-SCNlA plasmid was transfected into the N2a cell line,and the plasmid activity was verified by T7 endonuclease I(T7E1);? The pX459-SCN1 A plasmid was transfected into the HT22 cell line to construct a monoclonal cell line.The knockdown effect of SCN1A was detected by Western-Blot and QPCR.? The cell line with the highest knockout efficiency was selected as the SCN1A-KO group,and the WT group was used as a control.Transcriptome sequencing analysis was performed on total RNA from both cell lines.? Quality control of transcriptome raw data,comparison of reference genomes and calculation of differential genes.KEGG,GO enrichment and protein interaction network analysis were performed on differential genes.Partial differential genes were selected for QPCR validationResults ? Sequencing analysis of the constructed plasmid indicated that the designed three sgRNA was successfully inserted into the destination site,indicating that the plasmid was successfully constructed.? T7E1 digestion showed that the target fragment was mutated after transfection of pX459-SCN1A-2,indicating that pX459-SCN1A-2 has strong mutation activity?Western-Blot and QPCR are used to detect the knockdown effect of SCNIA.The results showed that pX459-SCN1A-2/1 had the highest knockout effect and the knockout efficiency exceeded 95%.? The pX45 9-SCN1A-2/1 cell line was used as the SCN1A-KO group,and the HT22 cells were used as a WT group for transcriptome sequencing analysis.The results showed that there were 1861 differential genes,of which 573 genes were up-regulated and 1288 genes were down-regulated.? The first five pathways of differential gene KEGG enrichment include tumor pathway,PI3-AKT signaling pathway,MAPK signaling pathway,HTLU-1 virus,human papillomavirus.The differential gene GO enrichment results indicated that differential gene enrichment is derived from the positive regulation of RNA polymerase ? promoter transcription,vascular remodeling,cytoplasmic nucleus,nuclear material,transcriptional regulation,DNA templated and other biological processes.Protein interaction network analysis showed that EHMT1,ACTA2,and MYC are the core of the protein network.And the ice fruit calculation obtained a core set of protein interactions.Related genes are involved in the process of RNA binding,splicing,processing and splicing of complexes.? Glycolysis-related genes such as PGM2,HK2,PFKL,PFKM,PGK1,and EN02 are down-regulated.Some of the calcium channel-related gene expression levels involved in cellular calcium homeostasis,calcium ion transport,guanylate cyclase,and second messenger-mediated signaling have changedConclusion Deprivation of Nay1.1 in neurons can lead to changes in various signaling pathways such as tumor pathway,PI3K-AKT signaling pathway,and MAPK signaling pathway,involving multiple biological processes such as transcription and translation of DNA Deprivation of neurons by Nay1.1 can cause down-regulation of glycolytic-related genes and disorder of calcium channel-related genes.Changes in MYC,EHMT1,and ACTA2 may play an important role in the pathogenesis of DS.