A new Friedreich ataxia (FA) experimental model generated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based gene editing of the social amoeba Dictyostelium discoideum may offer a new perspective on frataxin (FXN) activity and potential therapeutic solutions, according to an article preprint published in bioRxiv. This study has not yet been peer reviewed.
Before establishing D. discoideum as a model system for FA, the study authors tested the ability of D. discoideum FXN to substitute the human ortholog. They found the amoeba FXN capable of binding to the human supercomplex NFS1/ACP-ISD11/ISCU2 with a high affinity and effective at activating L-Cys desulfurase function.
The researchers then cultured the AX2 D. discoideum strain and edited its FXN locus using CRISPR/Cas9-mediated technology for rapid genome editing. They concluded that the FXN locus of the amoeba could be edited with no lethal consequences, producing a viable mutant strain with undetectable FXN expression levels.
The defective mutant strain was isolated as clone 8, presenting landmarks of FXN deficiency, including a decrease in iron-sulfur cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress.
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To confirm that all defects reported in clone 8 resulted from the lack of functional FXN, the authors expressed an intronless, C-terminal flag-tagged wild-type FXN version from a constitutive promoter. The experimental model responded with a full rescue of the growth defect but only a partial recovery of the decreased enzymatic activities.
“In this context, this new biological model offers a wide range of options to easily explore diverse phenotypes occurring in FA; this makes D. discoideum a very attractive approach for studying, in a straightforward manner, the effect that diversity of FA variants has on the cellular metabolism,” Gentili and colleagues wrote.
“Moreover, this model may help to understand whether FXN works as a bottleneck for some processes, whereas in other processes it has lower flux control, at least under the experimental conditions assayed.”
FXN, the main protein deficient in FA, is a key component of the mitochondrial supercomplex responsible for iron-sulfur cluster assembly. Several enzymatic reactions essential for cell homeostasis depend on iron-sulfur clusters.
Reference
Gentili HG, Pignataro MF, Olmos J, et al. CRISPR/Cas9-based edition of frataxin gene in Dictyostelium discoideum for Friedreich’s ataxia disease modeling. bioRxiv. Published online March 7, 2023. doi:10.1101/2023.02.27.530330
