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在指导RNA中的N1-甲基类伪尿素对CRISPR/Cas9活动的影响

Daria Prokhorova¹, Anastasiya Matveeva¹, Alexander Zakabunin¹

¹Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia.

International journal of molecular sciences

|December 9, 2023

概括

N1-methylpseudouridine (m1Ψ) 增强了CRISPR/Cas9基因组编辑,通过保留目标活动和减少目标外影响. 这种自然发生的RNA修饰显示了改善CRISPR/Cas9在体外和人体细胞中的应用的前景.

科学领域:

分子生物学分子生物学
生物化学生物化学
基因编辑技术的技术

背景情况:

通过引导RNA修改,CRISPR/Cas9基因编辑效率通常会得到提高.
虽然化学修饰得到了很好的研究,但自然发生的RNA修饰在很大程度上仍未被探索.
N1-methylpseudouridine (m1Ψ) 是一种天然存在的RNA修饰物,在mRNA疗法中已经得到了成熟的应用.

研究的目的:

调查N1-甲基伪尿素 (m1Ψ) 结合对CRISPR/Cas9系统功能的影响.
评估m1Ψ修饰导向RNA在增强CRISPR/Cas9活性和特异性的有效性.

主要方法:

采用FAM标记的dsDNA基质进行体外裂解试验,以确定修饰水平和动力参数.
计算m1Ψ修饰导向RNA的动力参数和特异性得分.
在哺乳动物细胞中评估经过修改的导向RNA活性,使用Neon转染和数字PCR.

主要成果:

m1Ψ 整合到指导RNA中可以保持目标基因组编辑效率.
在体外,N1-methylpseudouridine显著降低了CRISPR/Cas9的脱效应.
与m1Ψ修饰导向RNA配合的Cas9复合体在人类细胞中显示出有效的基因组编辑.

结论:

N1-methylpseudouridine是一种有希望的自然发生的RNA修饰,用于改进CRISPR/Cas9基因组编辑.
将m1Ψ纳入导向RNA可以增强CRISPR/Cas9的活性和特异性,无论是在体外还是在细胞环境中.
这种修改策略为推进CRISPR/Cas9治疗应用提供了一种有价值的方法.

关键词:

这就是CRISPR/Cas9的作用.这是一种N1-甲基pseudouridine.导向RNA的修改是指导RNA的修改.

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