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Generation of Leucine-Rich Repeat Kinase 2 (LRRK2) Knockout Neuroblastoma Cells SH-SY5Y by CRISPR/Cas9-Mediated
Hui-Lan Jong1, Kit-San Yuen2,3, Dong-Yan Jin2
1Department of Pre-Clinical Sciences, M. Kandiah Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Kajang, Selangor, Malaysia.
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.
Insights
Researchers developed a CRISPR/Cas9 strategy to effectively knockout low-expression Leucine-rich repeat kinase 2 (LRRK2) in Parkinson
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- Leucine-rich repeat kinase 2 (LRRK2) is implicated in Parkinson's disease pathogenesis.
- SH-SY5Y neuroblastoma cells, a common Parkinson's disease model, exhibit low LRRK2 expression, posing challenges for genetic manipulation.
- Understanding the role of low-expression genes like LRRK2 is crucial for disease research.
Purpose of the Study:
- To develop an effective strategy for knocking out low-expression Leucine-rich repeat kinase 2 (LRRK2) in SH-SY5Y cells.
- To overcome challenges associated with targeting low-expression genes using CRISPR/Cas9 technology.
- To establish a reliable LRRK2 knockout cell model for Parkinson's disease research.
Main Methods:
- Utilized a CRISPR/Cas9 system with a double-cut and multiple guide RNA strategy.
- Optimized electroporation parameters for enhanced plasmid delivery.
- Employed a refined clonal expansion technique and a sensitive protein detection protocol for validation.
Main Results:
- Successfully generated Leucine-rich repeat kinase 2 (LRRK2) knockout SH-SY5Y cells.
- Validated knockout efficiency through PCR analysis, DNA sequencing, and Western blot.
- Demonstrated the feasibility of targeting and knocking out low-expression genes in cell models.
Conclusions:
- The developed strategy enables effective knockout of low-expression Leucine-rich repeat kinase 2 (LRRK2) in SH-SY5Y cells.
- This provides a valuable tool for studying LRRK2's role in Parkinson's disease.
- The methodology can be applied to knockout other low-expression genes in various cellular contexts.
