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设计了IscB-ωRNA系统,用于基础编辑扩大了目标范围
Qingquan Xiao1,2, Guoling Li1, Dingyi Han2,3
1HuidaGene Therapeutics Co. Ltd., Shanghai, China.
Nature chemical biology
|August 15, 2024
概括
研究人员确定了新的IscB蛋白质用于基编辑,增强一个以扩大其准范围. 这些设计的IscB基编辑器对研究和治疗诸如杜申肌肉发育不良等遗传疾病有前途.
科学领域:
- 分子生物学分子生物学
- 基因编辑技术的技术
- 微生物基因组学 微生物基因组学
背景情况:
- IscB蛋白质是Cas9核酶的进化祖先,作为RNA引导的DNA内核酶和尼克酶起作用.
- 它们的紧性使得它们适合用于基础编辑应用,但有限的准范围限制了它们的实用性.
- 识别具有多种目标相邻动机 (TAM) 识别的IscB蛋白质对于扩展基编辑能力至关重要.
研究的目的:
- 在哺乳动物细胞中发现具有活性的新型IscB蛋白.
- 改造现有的 IscB 蛋白质以增强活性和扩大 TAM 识别.
- 开发IscB衍生基编辑器用于治疗应用,包括遗传疾病纠正.
主要方法:
- 从未培养的微生物中选非特征化的IscB蛋白质.
- 蛋白质和 ωRNA 工程来增强 IscB 活动和改变 TAM 特异性.
- 脱氨酶域与工程IscB尼克酶的融合,以创建基数编辑器.
- 在哺乳动物细胞和疾病模型中的基编辑器效率的体外和体内验证.
主要成果:
- 从19个未表征的微生物候选物中鉴定出10个活跃的IscB蛋白.
- 设计了IscB.m16以创建IscB.m16*,将TAM范围从MRNRAA扩展到NNNGNA.
- 开发了IscB.m16*衍生基编辑器,在哺乳动物细胞中表现出强大的效率.
- 在小鼠中使用单个AAV输送的基编辑器成功恢复了杜恩肌肉衰竭蛋白.
结论:
- 这项研究提供了一套来自新型和工程IscB蛋白质的紧基编辑工具.
- 扩大了IscB.m16*的TAM范围,提高了其用于基因编辑的多功能性.
- 基于IscB的基准编辑器对基因疾病的基础研究和治疗干预都有很大的潜力.
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