Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry⁺/EGFP⁺ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20–35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.

变化在叉头框G1（FOXG1）基因原因FOXG1综合征谱包括Rett综合征的先天性变体，其特征在于通过回归的早期发作，雷特状和急拉动作，和皮质视觉损伤。由于该转录调节子的损伤在下游主要是未知的病理生理机制，因此尚无具体的治疗方法。由于两个单倍剂量不足和过度表达FOXG1导致人类疾病的发生，我们认为在非天然调控序列下添加一个基因将是一种危险的策略，这与基因组编辑方法相反，在基因组编辑方法中，突变的基因被转化为野生型。在这里，我们证明了腺相关病毒（AAVs）偶联的CRISPR / Cas9系统能够靶向和纠正FOXG1源自患者的成纤维细胞，诱导多能干细胞（iPSC）和源自iPSC的神经元的变异。已经选择了变体特异性单向导RNA（sgRNA）和供体DNA，并与mCherry / EGFP报告系统一起克隆。将特定的sgRNA识别序列插入Cas9 CDS的上游和下游，以实现自我切割和失活。我们证明了AAV血清型的转导效率取决于靶细胞类型，其中最好的是成纤维细胞和iPSC衍生神经元中的AAV9，以及iPSC中的AAV2。mCherry ⁺ / EGFP ⁺的下一代测序（NGS）转染的细胞表明，在等位基因识别和脱靶活性方面，突变的等位基因均能高效修复（20–35％回复）和精确度。这项研究中测试的基因组编辑策略已被证明可以精确修复FOXG1，并且通过基于AAV9的系统进行递送代表了开发Rett综合征疗法的一步。