CRISPR/Cas9 and piggyBac-mediated footprint-free LRRK2-G2019S knock-in reveals neuronal complexity phenotypes and α-Synuclein modulation in dopaminergic neurons.
The p.G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) has been identified as the most prevalent genetic cause of familial and sporadic Parkinson's disease (PD). The Cre-LoxP recombination system has been used to correct the LRRK2-G2019S mutation in patient derived human induced pluripotent stem cells (hiPSCs) in order to generate isogenic controls. However, the remaining LoxP site can influence gene expression. In this study, we report the generation of a footprint-free LRRK2-G2019S isogenic hiPS cell line edited with the CRISPR/Cas9 and piggyBac technologies. We observed that the percentage of Tyrosine Hydroxylase (TH) positive neurons with a total neurite length of >2000μm was significantly reduced in LRRK2-G2019S dopaminergic (DA) neurons. The average branch number in LRRK2-G2019S DA neurons was also decreased. In addition, we have shown that in vitro TH positive neurons with a total neurite length of >2000μm were positive for Serine 129 phosphorylated (S129P) alpha-Synuclein (αS) and we hypothesize that S129P-αS plays a role in the maintenance or formation of long neurites. In summary, our footprint-free LRRK2-G2019S isogenic cell lines allow standardized, genetic background independent, in vitro PD modeling and provide new insights into the role of LRRK2-G2019S and S129P-αS in the pathogenesis of PD.
['Qing|Xiaobing|X|', 'Walter|Jonas|J|', 'Jarazo|Javier|J|', 'Arias-Fuenzalida|Jonathan|J|', 'Hillje|Anna-Lena|AL|', 'Schwamborn|Jens C|JC|']
D000369:Aged, 80 and over D064113:CRISPR-Cas Systems D002454:Cell Differentiation D002460:Cell Line D004251:DNA Transposable Elements D059290:Dopaminergic Neurons D005260:Female D005822:Genetic Vectors D006801:Humans D057026:Induced Pluripotent Stem Cells D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 D008297:Male D008875:Middle Aged D009154:Mutation D016501:Neurites D010641:Phenotype D010957:Plasmids D014162:Transfection D019895:Transposases D051844:alpha-Synuclein
CRISPR/Cas9 LRRK2-G2019S α-Synuclein Parkinson's disease hiPS piggyBac Serine129 phosphorylated α-Synuclein