A CRSIPR/dCas9-Targeted Histone Demethylation Induces GAA repeat contraction

弗里德里希共济失调(FRDA)是最常见的常染色体隐性遗传性神经肌肉疾病。这种疾病是 由Frataxin(FXN)基因第一内含子中扩大的GAA重复引起。目前尚无有效的治疗方法 由于患者基因组中剩余的扩大的重复序列，疾病是可用的。因此，一种治疗 迫切需要以扩大GAA重复为目标。我们发现，H3K9三甲基化的抑制 (H3K9me3)与DNA碱基切除修复(BER)协同收缩扩展的GAA重复序列和 上调FXN基因在FRDA神经细胞和转基因小鼠脑中的表达。我们假设GAA FXN基因H3K9me2/ME3重复靶向去甲基化可破坏异染色质并诱导BER 来收缩扩大的重复次数。为了验证这一假设，我们建议使用CRISPR/CAS9系统 组蛋白H3-三甲基-L-赖氨酸9去甲基酶4D与化脓性链球菌Cas9的融合 (CRISPR/dCas9-KDM4D)在FRDA神经细胞中诱导H3K9me2/ME3的GAA重复靶向去甲基化。 我们将追求两个具体目标。目的1是确定GAA重复序列靶向的CRISPR/dCas9-KDM4D是否可以 去甲基化H3K9me2/ME3以破坏FRDA神经细胞中FXN基因的异染色质。首先，我们将聚变 利用pCRISPR/dCas9-DNMT3A-PuroR_v2表达载体克隆人KDM4D基因与化脓性链球菌dCas9 作为主心骨。KDM4D将通过XTEN80连接器链连接到dCas9的C末端。这个 编码以5‘或3’侧翼区为靶点的单链引导RNA(SgRNAs)的序列 扩展的GAA重复序列也将被插入到该质粒中。该质粒将稳定地转入FRDA 从FRDA患者的诱导多能干细胞(IPSCs)中分化出的神经前体细胞(NPC)。 其次，我们将确定以重复序列为靶点的dCas9-KDM4D是否能够降低H3K9me2/ME3和 减轻从神经前体细胞分化的FRDA神经细胞中扩展重复序列的异染色质。目标2 目的是确定以GAA重复为靶点的CRISPR/dCas9-KDM4D是否通过 Ber，导致FXN基因表达上调，减轻线粒体功能障碍 FRDA神经细胞。首先，我们将确定dCas9-KDM4D是否会导致GAA重复收缩。到时候我们会的 确定dCas9-KDM4D是否可以促进关键的误码率酶，DNA聚合酶β(POLβ)， 和FEN1对FRDA神经细胞中扩展的重复序列的作用。第二，我们将测试dCas9- KDM4D可导致FXN基因表达上调，减轻线粒体功能障碍。我们的 这项研究将通过协同作用为扩大的GAA重复序列的基因靶向收缩提供概念证据 在组蛋白修饰和DNA修复之间。结果将揭示潜在的机制 CRISPR/dCas9-KDM4D通过组蛋白相互作用靶向收缩扩展的GAA重复序列 用BER去甲基化。本研究将进一步为FRDA的基因治疗开辟新的途径。

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 open a new avenue to develop effective gene therapy for FRDA.