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

弗里德赖希共济失调（FRDA）是最常见的常染色体隐性遗传性神经肌肉疾病。述疾病是 由共济失调蛋白（FXN）基因第一内含子中扩展的GAA重复序列引起。没有有效的治疗方法 由于患者基因组中保留了扩展的重复序列，因此可以获得疾病。因此，治疗， 因此，扩大GAA重复序列是迫切需要的。我们发现H3 K9三甲基化的抑制 （H3 K9 me 3）与DNA碱基切除修复（BER）协同收缩扩增的GAA重复序列， 在FRDA神经细胞和转基因小鼠脑中上调FXN基因表达。我们假设GAA FXN基因H3 K9 me 2/me 3的重复靶向去甲基化可破坏异染色质并诱导BER 以收缩扩展的重复序列。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脱甲基酶4D（KDM 4D）与催化失活的S.链球菌Cas9 在FRDA神经细胞中，使用CRISPR/dCas 9-KDM 4D来诱导H3 K9 me 2/me 3的GAA重复靶向去甲基化。 我们将追求两个具体目标。目的1是确定靶向GAA重复序列的CRISPR/dCas 9-KDM 4D是否可以 使H3 K9 me 2/me 3去甲基化以破坏FRDA神经细胞中FXN基因处的异染色质。首先，我们将融合 人KDM 4D基因与S.使用质粒pCRISPR/dCas 9-DNMT 3A-PuroR_v2 作为骨干。KDM 4D将通过XTEN 80接头链连接至dCas 9的C末端。的 用于编码靶向蛋白质的5 '或3'侧翼区的单链向导RNA（sgRNA）的序列。 扩增的GAA重复序列也将插入质粒中。质粒将稳定转染到FRDA中 从FRDA患者的诱导多能干细胞（iPSC）分化的神经祖细胞（NPC）。 第二，我们将确定重复靶向的dCas 9-KDM 4D是否可以降低H3 K9 me 2/me 3的水平，并且 减轻从NPC分化的FRDA神经细胞中扩增重复序列的异染色质化。目的2 目的是确定靶向GAA重复序列的CRISPR/dCas 9-KDM 4D是否通过以下途径促进GAA重复序列收缩： BER，导致FXN基因表达的上调和线粒体功能障碍的缓解， FRDA神经细胞首先，我们将确定dCas 9-KDM 4D是否可以导致GAA重复收缩。然后我们将 确定dCas 9-KDM 4D是否可以促进关键BER酶DNA聚合酶β（Pol β）的募集， 和侧翼核酸内切酶1（FEN 1）与FRDA神经细胞中扩增的重复序列的关系。第二，我们将测试dCas 9- KDM 4D可导致FXN基因表达上调并减轻线粒体功能障碍。我们 这项研究将为通过协同作用使扩增的GAA重复序列发生基因靶向收缩提供概念证明 组蛋白修饰和DNA修复之间的联系结果将揭示潜在的机制 CRISPR/dCas 9-KDM 4D通过组蛋白的相互作用靶向收缩扩展的GAA重复序列 用BER进行脱甲基。该研究将进一步为FRDA的有效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.