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减少基RNA内部固有的自身抑制相互作用,提高了主要编辑效率

Karthikeyan Ponnienselvan¹, Pengpeng Liu¹, Thomas Nyalile²

¹Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.

Nucleic acids research

|May 29, 2023

概括

优化原始编辑 (PE) 涉及调整 pegRNAs 中的原始结合点 (PBS) 长度以提高效率. 这项研究完善了PE系统,用于在没有双链断裂的情况下精确编辑基因组,从而改善治疗应用.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
基因组学就是基因组学.

背景情况:

主编辑能够在没有双链断裂的情况下精确修改基因组.
之前的研究表明,对于主要编辑指导RNA (pegRNAs) 来说,最优的原始结合位点 (PBS) 长度为~13个核酸.
最佳PBS长度表征以前仅限于等离子体或lentiviral表达系统.

研究的目的:

调查PBS-spacer互补性对主要编辑器 (PE) 核糖蛋白 (RNP) 复合物的效率的影响.
用PE-RNP系统来确定最佳的pegRNA设计参数,以增强使用PE-RNP系统的原始编辑结果.
在细胞模型和体内验证精细的PE-pegRNA设计.

主要方法:

在pegRNAs中,PBS和间隔序列之间的自抑制相互作用的表征.
系统地改变PBS长度和互补性,以优化pegRNA结合和目标识别.
在哺乳动物细胞中使用末端受保护的pegRNAs评估主要编辑效率.
暂时冷冲击处理的应用,以增强主要编辑结果.
在患者衍生纤维细胞,原始人类T细胞和斑马鱼中验证优化的PE-RNP复合体.

主要成果:

减少PBS-spacer区域之间的互补性通过破坏自我抑制相互作用来提高主要编辑效率.
对于末端受保护的pegRNAs,在哺乳动物细胞中,较短的PBS长度,化温度接近37°C是最佳的.
在分娩后的过渡冷冲击治疗进一步提高了原始编辑效率.
优化的PE-RNP复合体成功地纠正了与疾病相关的突变,并在各种细胞类型和生物体中安装了精确的编辑.

结论:

精细的pegRNA设计参数,包括优化的PBS长度和减少的互补性,显著提高了PE-RNP的主要编辑效率.
这些发现为治疗应用和遗传研究提供了更强大,更有效的原始编辑系统.
这项研究提升了原始编辑技术的精度和适用性,用于纠正遗传缺陷和工程基因组.

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