Inherited retinal dystrophies (IRDs) are characterized by progressive photoreceptor degeneration and vision loss. Usher syndrome (USH) is a syndromic IRD characterized by retinitis pigmentosa (RP) and hearing loss. USH is clinically and genetically heterogeneous, and the most prevalent causative gene is USH2A. USH2A mutations also account for a large number of isolated autosomal recessive RP (arRP) cases. This high prevalence is due to two recurrent USH2A mutations, c.2276G>T and c.2299delG. Due to the large size of the USH2A cDNA, gene augmentation therapy is inaccessible. However, CRISPR/Cas9-mediated genome editing is a viable alternative. We used enhanced specificity Cas9 of Streptococcus pyogenes (eSpCas9) to successfully achieve seamless correction of the two most prevalent USH2A mutations in induced pluripotent stem cells (iPSCs) of patients with USH or arRP. Our results highlight features that promote high target efficacy and specificity of eSpCas9. Consistently, we did not identify any off-target mutagenesis in the corrected iPSCs, which also retained pluripotency and genetic stability. Furthermore, analysis of USH2A expression unexpectedly identified aberrant mRNA levels associated with the c.2276G>T and c.2299delG mutations that were reverted following correction. Taken together, our efficient CRISPR/Cas9-mediated strategy for USH2A mutation correction brings hope for a potential treatment for USH and arRP patients.

中文翻译：

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患者iPSC中的基因组编辑可纠正最普遍的USH2A突变，并揭示出令人感兴趣的突变mRNA表达谱。

遗传性视网膜营养不良（IRD）的特征是进行性感光细胞变性和视力丧失。Usher综合征（USH）是一种综合症IRD，其特征在于色素性视网膜炎（RP）和听力丧失。USH在临床和遗传上是异质的，最普遍的致病基因是USH2A。USH2A突变也占了大量孤立的常染色体隐性RP（arRP）病例。这种高患病率是由于两个反复出现的USH2A突变，即c.2276G> T和c.2299delG。由于USH2A cDNA的大小较大，因此无法进行基因增强疗法。但是，CRISPR / Cas9介导的基因组编辑是一种可行的选择。我们使用增强的化脓链球菌Cas9（eSpCas9）特异性Cas9成功实现了USH或arRP患者的诱导多能干细胞（iPSC）中两个最普遍的USH2A突变的无缝校正。我们的结果突出了可促进eSpCas9的高靶标疗效和特异性的功能。一致地，我们没有在校正的iPSC中发现任何脱靶诱变，这也保留了多能性和遗传稳定性。此外，对USH2A表达的分析出乎意料地确定了与c.2276G> T和c.2299delG突变相关的异常mRNA水平，这些突变在校正后已恢复。综上所述，我们有效的CRISPR / Cas9介导的USH2A突变校正策略为USH和arRP患者的潜在治疗带来了希望。我们的结果突出了可促进eSpCas9的高靶标疗效和特异性的功能。一致地，我们没有在校正的iPSC中发现任何脱靶诱变，这也保留了多能性和遗传稳定性。此外，对USH2A表达的分析出乎意料地确定了与c.2276G> T和c.2299delG突变相关的异常mRNA水平，这些突变在校正后已恢复。综上所述，我们有效的CRISPR / Cas9介导的USH2A突变校正策略为USH和arRP患者的潜在治疗带来了希望。我们的结果突出了可促进eSpCas9的高靶标疗效和特异性的功能。一致地，我们在校正后的iPSC中未发现任何脱靶诱变，这也保留了多能性和遗传稳定性。此外，对USH2A表达的分析出乎意料地确定了与c.2276G> T和c.2299delG突变相关的异常mRNA水平，这些突变在校正后已恢复。综上所述，我们有效的CRISPR / Cas9介导的USH2A突变校正策略为USH和arRP患者的潜在治疗带来了希望。对USH2A表达的分析出乎意料地确定了与c.2276G> T和c.2299delG突变相关的异常mRNA水平，这些突变在校正后得以恢复。综上所述，我们有效的CRISPR / Cas9介导的USH2A突变校正策略为USH和arRP患者的潜在治疗带来了希望。对USH2A表达的分析出乎意料地确定了与c.2276G> T和c.2299delG突变相关的异常mRNA水平，这些突变在校正后得以恢复。综上所述，我们有效的CRISPR / Cas9介导的USH2A突变校正策略为USH和arRP患者的潜在治疗带来了希望。