为CRISPR干扰/激活 (CRISPRi/a) 应用提供dCas9的稳健表达和单步净化的一种新方法
Harshita Pandey1, Binduma Yadav1, Koushik Shah2
1National Agri-Food Biotechnology Institute, Knowledge City, Sector 81, Mohali, Punjab, 140306, India; Regional Center for Biotechnology, Faridabad, Haryana, 160014, India.
Protein expression and purification
|May 8, 2024
概括
使用大肠杆菌 (E. coli) 开发了一种简化的一步净化方法,用于失活的Cas9 (dCas9) 蛋白. 这种高效的协议可以为各种基因组编辑和基因调节应用产生纯 dCas9.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 基因组学就是基因组学.
背景情况:
- CRISPR-Cas9是一种多功能基因组编辑工具.
- 失活的Cas9 (dCas9) 能够调节基因表达 (CRISPRa/CRISPRi) 和在没有DNA裂变的情况下进行相互作用研究.
研究的目的:
- 开发一种强大而高效的方法来生产纯的dCas9蛋白质.
- 为了促进dCas9.9的结构和功能表征.
主要方法:
- 在大肠杆菌宿主中使用N端酸性标签表达dCas9.
- 使用廉价的多模态酸染色学进行单步净化.
主要成果:
- 为高产量dCas9生产建立了一个简单的单步协议.
- 纯化的dCas9蛋白质是均的,适合各种应用.
结论:
- 开发的方法提供了一种获得纯dCas9.9的有效方法.
- 这种纯化的dCas9对体外和体内基因编辑和调节的研究有价值.
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