Prime editing is a hybrid genome editing technology that introduces small edits on the genome with high precision. It combines nickase Cas9 with reverse transcriptase to prime and synthesizes edited DNA from RNA, reducing unintended insertions and deletions on the genome. This protocol describes the design of prime editing guide RNAs (pegRNAs), cloning of plasmids, nucleofection of mouse primary neurons, and preparation for next-generation sequencing. Directions are given for pegRNA and PE3b gRNA design and construction using PegAssist, a publicly available webtool and plasmid set. Prime editing in neurons allows genome manipulation while maintaining endogenous gene expression, making it ideal for studying protein structure/function relationships and pathogenic variants in a native neuronal context.