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对CRISPR-Cas13bRNA基编辑方法的比较,用于USH2A相关的遗传视网膜退化

Lewis E Fry^1,2,3, Lauren Major¹, Ahmed Salman¹

¹Nuffield Department of Clinical Neurosciences & NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, UK.

Communications biology

|February 8, 2025

概括

在CRISPR-Cas13RNA编辑成功地修复了USH2A基因突变导致阿舍尔综合征在培养细胞和小鼠模型. 这表明了转录组向基因疗法的潜力,用于遗传性视网膜疾病.

科学领域:

分子生物学分子生物学
基因治疗基因治疗
眼科医生眼科眼科

背景情况:

克里斯普尔-Cas13系统为精确的RNA点突变纠正提供了潜力.
在RNA (ADAR) 效应器上作用的腺氨酸脱氨酶能够实现有针对性的A-to-I基转换.
USH2A基因突变是遗传视网膜疾病和阿舍尔综合征的常见原因.

研究的目的:

为了比较Cas13效应器 (PspCas13b和Cas13bt3) 修复USH2A基因突变的疗效.
开发和描述一种小鼠模型,用于评估遗传性视网膜疾病中的基编辑器.
评估转录组向基因编辑对视网膜疾病的治疗潜力.

主要方法:

用各种导向RNA (gRNA) 和促进剂在培养细胞中进行USH2A基因修复的PspCas13b和Cas13bt3因子的比较.
一种具有特定USH2A突变 (c.11840 G>A) 的阿舍尔综合征小鼠模型的开发和表征.
在体内评估通过腺相关病毒 (AAV) 传递到小鼠光受体中的Cas13效应器.

主要成果:

在培养细胞中修复USH2A突变 (c.11864 G>A和c.11840 G>A) 的效率高达80%.
光受体中的活体RNA编辑率在0.32%至2.04%之间,其中PspCas13b显示出比Cas13bt3.3.b更高的效率.
用PspCas13b构造物治疗的小鼠在连接膜中恢复和正确定位乌舍林蛋白.

结论:

PspCas13b和Cas13bt3是有效的Cas13因子,可以在RNA水平上纠正USH2A突变.
开发的小鼠模型适用于评估遗传性视网膜疾病的基准编辑器.
转录基因组向基因编辑对治疗视网膜疾病如阿舍尔综合征具有前途.

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