Abstract
Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1–3% of Parkinson’s disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.
Highlights
Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1–3% of Parkinson’s disease (PD) cases worldwide
A guide RNA was designed so that the 20-nucleotide variable domain was homologous to the g.G6055 site, and compatible with a protospacer adjacent motif (PAM) (Fig. 1b)
The top eight loci that were predicted to be potential off-target sites for the guide RNA (gRNA) were individually sequenced in two homozygous LRRK2 G2019S clones derived from both Cj-ESCs and Cj-iPSCs (Supplemental Table 1)
Summary
Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1–3% of Parkinson’s disease (PD) cases worldwide. We report our successful use of CRISPR/Cas[9] with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. These phenotypes were not observed in clones with LRRK2 truncation These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S. Phenotypic evaluation of dopaminergic neurons derived from the mutant marmoset stem cell lines showed LRRK2 G2019S-mediated changes in cell homeostasis and neurite branching These methods will aid in the development of novel genetic-based models of PD LRRK2 G2019S, as well as new gene targets in marmosets and other nonhuman primate species
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