CRISPR/Cas9-mediated genetic correction reverses spinocerebellar ataxia 3 disease-associated phenotypes in differentiated cerebellar neurons

Abstract

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3; also called Machado-Joseph disease, MJD) is a trinucleotide repeat disorder caused by expansion of the CAG repeats in the ATXN3 gene. Here, we applied a CRISPR/Cas9-mediated approach using homologous recombination to achieve a one-step genetic correction in SCA3-specific induced pluripotent stem cells (iPSCs). The genetic correction reversed disease-associated phenotypes during cerebellar region-specific differentiation. In addition, we observed spontaneous ataxin-3 aggregates specifically in mature cerebellar neurons differentiated from SCA3 iPSCs rather than in SCA3 pan-neurons, SCA3 iPSCs or neural stem cells, suggesting that SCA3 iPSC-derived disease-specific and region-specific cerebellar neurons can provide unique cellular models for studying SCA3 pathogenesis in vitro. Importantly, the genetically corrected cerebellar neurons did not display typical SCA3 aggregates, suggesting that genetic correction can subsequently reverse SCA3 disease progression. Our strategy can be applied to other trinucleotide repeat disorders to facilitate disease modeling, mechanistic studies and drug discovery.

Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system

“…We speculated that novel Acr-based controllable tools for a wide range of applications can be developed using the PICI system with broad-spectrum inhibitors such as AcrIIC1 and AcrIIA5 ( 26 , 27 ). In particular, developing more advanced Acr-based tools to achieve precise, allele-specific and controllable genome editing may provide new opportunities for biomedical applications in the future ( 49 , 50 ).…”

4E-BP1 counteracts human mesenchymal stem cell senescence via maintaining mitochondrial homeostasis

“…CRISPR/Cas9-mediated gene editing was performed as previously described ( Chu et al , 2022 ). In brief, sgRNA targeting exon 1 of EIF4EBP1 was designed using an online system as previously reported ( Song et al , 2022 ), and then cloned into pCAG-mCherry-sgRNA vector (Addgene, #87110). EIF4EBP1 +/+ hESCs cultured on Matrigel-coated plates (Corning) were treated with ROCK inhibitor (Y-27632, TOCRIS) for 10 h before electroporation.…”

A narrative review of organoids for investigating organ aging: opportunities and challenges

“…[130] IPSCs-derived cerebellar organoid of spinocerebellar ataxia type 3 (SCA3), the second most common polyglutamine disease after Huntington's disease, has been observed spontaneous ataxin-3 aggregates in neurons, and the SCA3 disease-associated phenotypic abnormalities in cerebellar neurons can be reversed by the genetic correction. [131] With the advances in organoid technologies, we are optimistic that the use of brain organoids can provide us with valuable knowledge of molecular mechanisms underlying NDDs and illuminate novel ideas for developing targeted therapy.…”