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优化Cas12a用于多重基因组规模的转录激活

Audrey L Griffith¹, Fengyi Zheng¹, Abby V McGee¹

¹Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA.

|September 18, 2023

概括

这项研究引入了一种新的Cas12aCRISPR激活 (CRISPRa) 系统,用于人类细胞中的多重基因激活. 新系统可以同时激活多达四个基因,加速基因选.

科学领域:

分子生物学分子生物学
遗传学遗传学是一个
生物技术是生物技术.

背景情况:

Cas12a的CRISPR技术提供了导向RNA的轻松复合,有利于组合扰动.
现有的Cas12a应用主要用于淘汰屏幕;需要对人类细胞中的CRISPR激活 (CRISPRa) 进行优化.

研究的目的:

开发和验证一种基于Cas12a的新型CRISPR激活 (CRISPRa) 系统,用于人类细胞中的多重基因激活.
通过全基因组CRISPRa屏幕识别细胞生长和MEK抑制的调节剂.

主要方法:

开发一个基于Cas12a的Transactivation Domain (TAD) 招聘系统,使用ALFA纳米体.
在人类细胞中进行全基因组CRISPRa查,以确定生长调节剂和MEK抑制.
基于Cas12a的CRISPRa结果与开放式读取框架 (ORF) 过度表达和基于Cas9的CRISPRa结果的比较.

主要成果:

使用开发的Cas12a-CRISPRa系统,证明了多达四个基因的同时激活.
确定了增长和MEK抑制的关键调节剂.
发现多重阵列活动可以从表现最好的单向导RNA中预测.

结论:

开发的基于Cas12a的CRISPRa系统能够在人类细胞中有效地激活多重基因.
这项技术显著加速了基因功能和组合性表型在规模上的探索.
提供了在多重CRISPRa屏幕中选择主动指南的标准.

关键词:

在CRISPR中激活CRISPR.这就是Cas12a.功能性基因组学功能性基因组学遗传选器遗传选器

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