Principal Investigator University of California, Berkeley Berkeley, California, United States
Abstract: Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer a unique experimental platform to advance our understanding of PD etiology by enabling the generation of disease-relevant cell-types carrying patient mutations along with isogenic control cells. To facilitate this approach, we generated a collection of over 60 cell lines genetically engineered to harbor high risk or causal variants in genes associated with PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G+FS, SYNJ1 R258Q/FS, VPS13C A444P, VPS13C W395C, GBA1 IVS2+1). All mutations were generated in a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using CRISPR/Cas9 or prime editing-based approaches. We implemented rigorous quality controls, including whole genome sequencing of each cell line. Our analysis of the genetic variation in this cell line collection revealed that while genome editing, particularly using CRISPR/Cas9, can introduce rare off-target mutations, the predominant source of genetic variants arises from routine cell culture. While perfect isogeneity remains elusive, the observed genetic variation was minimal compared to those typically found in patient-derived iPSCs, predominantly affecting non-coding regions of the genome. Importantly, the genetic variation can be effectively managed through stringent quality control measures and careful experimental design. This systematic approach ensures the high quality of our stem cell collection, highlights differences between conventional CRISPR/Cas9 and prime editing and provides a roadmap for how to generate gene-edited hPSCs collections at scale in an academic setting. Our iSCORE-PD collection represents an easily accessible and valuable platform to study PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.
