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开发使用高通量测量的紧的转录效应器,在不同的环境中进行高通量测量

Josh Tycko^1,2, Mike V Van³, Aradhana¹

¹Department of Genetics, Stanford University, Stanford, CA, USA.

Nature biotechnology

|November 2, 2024

概括

研究人员系统地选了蛋白质领域的基因调节效应,发现了取决于背景的功能. 他们开发了改进的CRISPR工具,用于基因沉默和激活,增强了CAR T细胞治疗等应用.

科学领域:

分子生物学分子生物学
基因组学就是基因组学.
表观遗传学在表观遗传学中,表观遗传学是指表观遗传学.

背景情况:

转录效应器调节基因表达,但它们在不同的基因组和细胞环境中的功能尚未完全理解.
需要有系统的理解来利用这些领域来准确调节基因.

研究的目的:

系统量化核蛋白域在调节跨多种基因组和细胞类型背景的转录中的功能.
为了确定环境强大的效应因子域,以改进基于CRISPR的基因编辑工具.
为增强CRISPR激活 (CRISPRa) 应用设计新型激活剂.

主要方法:

开发了一种使用dCas9的高通量招募 (HT-recruit) 选方法,以量化内源基因的效应器功能.
测试了5,092个核蛋白Pfam域的库和一个更大的未注释区域库.
选择了具有环境稳定的域名和设计了新的CRISPR激活器.

主要成果:

许多转录效应因子域表现出上下文依赖的活动,根据目标和DNA结合域 (DBD) 语境作为激活器或抑制器起作用.
识别了诸如ZNF705 KRAB之类的环境强大的域,用于改进CRISPR干扰 (CRISPRi) 基因沉默.
通过结合NCOA3,FOXO3和ZNF473域,为高效的CRISPRa.设计了一种新的紧型人类激活器 (NFZ).

结论:

系统查揭示了特定于环境的转录效应因子功能,这对于理解基因调节至关重要.
开发了改进的CRISPRi和CRISPRa工具,提高了效率,病毒传递和可诱导控制,用于治疗应用,如CAR T细胞工程.

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