基因组规模的转录激活由一个工程 CRISPR-Cas9 复合体
Silvana Konermann1, Mark D Brigham1, Alexandro E Trevino1
11] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Nature
|December 11, 2014
概括
工程CRISPR-Cas9激活器使得功能研究的基因表达精确控制. 这项技术使大规模的屏幕能够识别赋予药物耐药性的基因,从而推动了基因扰乱研究.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 强大的基因表达扰动对于理解基因功能至关重要.
- 克里斯普尔-Cas9技术为基因操纵提供了一个强大的平台.
研究的目的:
- 设计一个CRISPR-Cas9复合体,以在内生基因组位点进行高效的转录激活.
- 为了研究转录激活的单导向RNA (sgRNA) 向规则.
- 选激活后对BRAF抑制剂产生抗性的基因.
主要方法:
- 用于转录激活的CRISPR-Cas9复合体的结构引导工程.
- 利用工程 Cas9 激活复合体来研究 sgRNA 向规则.
- 执行多重基因激活和高调长跨基因非编码RNA (lincRNA) 转录.
- 合成了大量的指南库,用于针对BRAF抑制剂耐药性的全基因组屏幕.
主要成果:
- 通过使用工程 Cas9 激活器,在内源基位表现出高效的转录激活.
- 确定了有效转录激活的sgRNA向规则.
- 成功实现了十个基因的多重激活和上调的lincRNA转录.
- 发现了已知和新型基因,通过大规模屏幕赋予BRAF抑制剂耐药性.
- 验证了查结果,并发现了与BRAF抑制剂耐药性相关的基因表达特征.
结论:
- 工程 Cas9 激活器代表了一种用于基因扰乱的强大技术.
- 这种方法可以实现高效的转录激活和大规模的功能性基因组屏幕.
- 这些发现对理解耐药性机制和开发新的治疗策略具有重要意义.
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