Friedreich’s Ataxia (FRDA) is the most common autosomal recessive neuromuscular disorder. The disease is caused by expanded GAA repeats in the first intron of the frataxin (FXN) gene. No effective treatments for the disease are available, owing to the expanded repeats remaining in the patients’ genome. Thus, a treatment that targets the expanded GAA repeats is urgently needed. We found that the inhibition of H3K9 trimethylation (H3K9me3) synergized with DNA base excision repair (BER) to contract the expanded GAA repeats and upregulate FXN gene expression in FRDA neural cells and transgenic mouse brain. We hypothesize that GAA repeat-targeted demethylation of H3K9me2/me3 at the FXN gene can disrupt heterochromatin and induce BER to contract the expanded repeats. To test this hypothesis, we propose to use a CRISPR/Cas9 system with the histone H3-trimethyl-L-Lysine 9 demethylase 4D (KDM4D) fused to catalytically inactivated S. pyogenes Cas9 (CRISPR/dCas9-KDM4D) to induce GAA repeat-targeted demethylation of H3K9me2/me3 in FRDA neural cells. We will pursue two Specific Aims. Aim 1 is to determine if the GAA repeat-targeted CRISPR/dCas9-KDM4D can demethylate H3K9me2/me3 to disrupt heterochromatin at the FXN gene in FRDA neural cells. First, we will fuse the human KDM4D gene with the S. pyogenes dCas9 using the plasmid pCRISPR/dCas9-DNMT3A-PuroR_v2 as a backbone. KDM4D will be linked to the C-terminus of dCas9 through the XTEN80 linker chain. The sequences for coding the single-strand guide RNAs (sgRNAs) that target the 5’- or 3’-flanking regions of the expanded GAA repeats will also be inserted into the plasmid. The plasmid will be stably transfected into FRDA neural progenitor cells (NPCs) differentiated from induced pluripotent stem cells (iPSCs) of an FRDA patient. Second, we will determine if the repeat-targeted dCas9-KDM4D can reduce the level of H3K9me2/me3 and alleviate heterochromatinization on the expanded repeats in FRDA neural cells differentiated from NPCs. Aim 2 is to determine if the GAA repeat-targeted CRISPR/dCas9-KDM4D promotes GAA repeat contraction through BER, leading to the upregulation of the FXN gene expression and the alleviation of mitochondrial dysfunction in FRDA neural cells. First, we will determine if dCas9-KDM4D can lead to GAA repeat contraction. We will then determine if dCas9-KDM4D can facilitate the recruitment of the key BER enzymes, DNA polymerase β (Pol β), and flap endonuclease 1 (FEN1) to the expanded repeats in FRDA neural cells. Second, we will test if dCas9- KDM4D can result in the upregulation of the FXN gene expression and alleviate mitochondrial dysfunction. Our study will provide proof of concept for a gene-targeted contraction of expanded GAA repeats via the synergy between histone modifications and DNA repair. The results will reveal the mechanisms underlying CRISPR/dCas9-KDM4D targeted contractions of expanded GAA repeats through the interplay of histone demethylation with BER. The study will further o...