通过减少错误折叠的pegRNA相互作用来增强CRISPR主要编辑.
Weiting Zhang1,2, Karl Petri3,4, Junyan Ma1,2,5
1Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.
bioRxiv : the preprint server for biology
|August 30, 2023
概括
在主要编辑指导RNA (pegRNAs) 中的内部序列互补性可以降低CRISPR主要编辑 (PE) 的效率. 一个简单的pegRNA重新折叠程序显著提高了斑马鱼胚胎的PE效率.
科学领域:
- 分子生物学分子生物学
- 基因编辑技术的技术
背景情况:
- 克里斯普尔主要编辑 (PE) 使用一个Cas9尼克酶-逆转录酶融合蛋白 (PE2) 和一个主要编辑指导RNA (pegRNA).
- RNA是扩展导向RNA,编码目标特异性和所需的遗传编辑.
- RNA内部的内部序列互补性可以阻碍Cas9复合并降低PE效率.
研究的目的:
- 调查pEGRNA内部内部序列互补性对PE效率的影响.
- 为了确定克服由pegRNA结构引起的降低PE效率的策略.
- 为了提高主要编辑技术的有效性.
主要方法:
- 在pegRNAs中对序列互补性的分析.
- 开发和应用PegRNA重新折叠程序.
- 引入点突变来破坏内部pegRNA相互作用.
- 在斑马鱼胚胎中评估PE效率.
主要成果:
- RNAs的5'和3'区域之间的序列互补性会对Cas9复杂化和PE效率产生负面影响.
- 一种简单的pegRNA重新折叠程序在斑马鱼胚胎中提高了核蛋白介导的PE效率高达25倍.
- 引入特定的点突变进一步提高了PE效率,高达6倍.
结论:
- 内部序列互补性是限制RNA功能的关键因素.
- 佩格RNA重新折叠和向突变策略提供了简单但有效的方法来提高CRISPR主要编辑效率.
- 这些发现为优化PE提供了切实可行的方法,用于基因组工程中的更广泛应用.
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