通过CRISPRRNA架构的同时多功能转录组工程
Zukai Liu1,2, Nathaniel Jillette1, Paul Robson1,2,3,4
1The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA.
Nucleic acids research
|July 3, 2023
概括
一个名为Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST) 的新平台可以同时操纵多个RNA目标. 这种系统显著减少了非目标效应,提高了RNA工程能力.
科学领域:
- 分子生物学分子生物学
- 在RNA生物学,RNA生物学.
- 生物技术是生物技术.
背景情况:
- 精确调节RNA处理和新陈代谢对于细胞完整性和功能至关重要.
- 克里斯普尔-Cas13系统允许有针对性的RNA工程,但同时调制多个RNA处理步骤和非目标效应仍然是挑战.
研究的目的:
- 开发一种用于同时,多功能RNA调制的新平台.
- 克服现有的RNA工程工具的局限性,包括非目标事件.
主要方法:
- 通过Scaffold开发了组合RNA工程 Tagged gRNA (CREST) 平台.
- 将附加的RNA支架连接到Cas13 gRNA和与酶域结合的RNA结合蛋白.
- 创建了用于RNA替代拼接和基编辑的双功能和三功能CREST系统 (A-to-G,C-to-U).
- 使用分裂设计方法进行酶复制,以最大限度地减少目标之外的事件.
主要成果:
- 证明了替代拼接和基基编辑的同时RNA操纵.
- 使用分裂酶设计,实现了非目标事件的显著减少 (近99%).
- 在目标部位成功复制了酶活性.
结论:
- CREST平台可以同时执行多个RNA调制函数在不同的RNA目标上.
- 分拆设计策略有效地减少了目标外影响,提高了特异性.
- CREST扩展了用于RNA生物学研究的转录组工程工具箱.
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