Construction Of Munc13-1 Gene Knockout Neuro-2a Cell Line With CRISPR/Cas9 System

The mechanism of neurotransmitter release involves a series of protein and protein interactions.Ca²⁺-triggered synaptic vesicle fusion mediated by SNARE complex assembly occupy the key status.Munc13-1 performs an important role in regulating the synaptic vesicle fusion process.In order to clarify the mechanism of Munc13-1 in neurosynaptic vesicle priming,Munc13-1 gene knockout neuroblastoma?Neuro-2a?cell lines were constructed by using clustered regularly interspaced short palindromic repeats/Cas9 nuclease?CRISPR/Cas9?gene editing system.The knockout of Munc13-1gene mediated by Cas9 nuclease targeting to specific genomic loci with single-guide RNA?sgRNA?sequence,which leads to certain mismatches of the DNA and thereby promotes mutagenesis in Neuro-2a cells.In this study,a series of experiments were performed and the results were as follows:Three sgRNAs were designed for Munc13-1 gene knockout according to Munc13-1 29^thh exon?Number:ENSMUSE00000329984?.Three sgRNAs were constructed into CRISPR/Cas9 system plasmids PX458 and PX459,which could be screened by GFP or puromycin respectly.The PX459-sgRNA constructions were transfected to Neuro-2a cells and the sgRNA2 of higher activity was indentified by sequence analysis.The PX458-sgRNA2 construction was transfected to Neuro-2a cells,and the Munc13-1 gene knockout Neuro-2a cell line was obtained by flow cytometry sorting and limited dilution.Munc13-1 gene knockout Neruro-2a cell line was identified by testing the form of insertion/deletion?indel?mutations and protein expression level.Munc13-1 gene knockout Neuro-2a cell line was constructed preliminarily by CRISPR/Cas9 system in this study,paved the way for further exploring the molecular mechanism of Munc13-1 in neurosynaptic vesicle seretion.