Generation of Leucine-Rich Repeat Kinase 2 (LRRK2) Knockout Neuroblastoma Cells SH-SY5Y by CRISPR/Cas9-Mediated Genome Editing

Abstract

Leucine-rich repeat kinase 2 (LRRK2) is associated with Parkinson’s disease, despite its low expression in the brain. Pathogenic mutations in LRRK2 enhance kinase activity and contribute to the disease’s pathogenesis. Neuroblastoma SH-SY5Y cells, which also exhibit low LRRK2 expression, are extensively used as a model for Parkinson’s disease. While less prominent, low-expression genes can play crucial roles in cellular processes, development, and disease. Knocking out such genes poses specific challenges, including difficulties in detection, incomplete knockout, and compensatory mechanisms that can obscure phenotypic changes. This study develops a strategy to knockout low-expression LRRK2 in SH-SY5Y cells effectively. Our approach employs a double-cut and multiple guide RNAs strategy, optimized electroporation parameters to enhance CRISPR/Cas9 plasmid delivery, refined clonal expansion technique, and a sensitive protein detection protocol. We successfully generate LRRK2 knockout SH-SY5Y cells using CRISPR/Cas9, with the knockout efficiency validated by PCR analysis, sequencing, and Western blot analysis.