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建立可诱导CRISPR干扰系统的协议,用于在人类多能干细胞中对多个基因进行沉默

Satoshi Matsui¹, Joseph R Shiley¹, Marissa Granitto¹

¹Division of Developmental Biology, Center for Stem Cell & Organoid Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, USA.

|July 31, 2024

概括

这项研究引入了一种可诱导多西环素的CRISPR干扰 (CRISPRi) 系统,以有效地静止人类诱导多能干细胞 (hPSC) 中的多个基因. 该协议简化了复杂基因淘汰模型的创建,节省了时间和资源.

科学领域:

干细胞生物学干细胞生物学
基因编辑技术的技术
分子遗传学分子遗传学

背景情况:

诱导性功能丧失策略对于剖析基因功能至关重要.
在人类诱导多能干细胞 (hPSCs) 中开发可诱导的多基因淘汰模型是复杂而劳动密集的.

研究的目的:

提出一个建立可诱导多克西环素的CRISPR干扰 (CRISPRi) 系统的协议.
为了使hPSCs中的多个基因同时沉默.
为了克服与创建可诱导,多基因淘汰模型相关的挑战.

主要方法:

主机CRISPRi hPSCs的建立.
设计和单导向RNAs (sgRNAs) 的lentiviral克隆.
产生和选择单克隆CRISPRi hPSC线条.
识别有效的CRISPRi克隆的程序.

主要成果:

在hPSC中成功建立了可诱导多克西环素的CRISPRi系统.
证明了多个基因的同时沉默.
创建和验证可诱导多基因淘汰hPSC线路的详细协议.

结论:

开发的CRISPRi系统提供了一种可诱导的高效方法,用于hPSC中的多基因沉默.
该协议简化了用于功能基因组学研究的复杂遗传模型的生成.
该系统有助于更深入地了解人类多能干细胞中的基因功能.

关键词:

克里斯普尔是什么意思?克里斯普尔是什么意思?细胞生物学细胞生物学细胞分化细胞分化细胞培养培养的细胞培养.细胞隔离细胞的隔离.分子生物学分子生物学干细胞是干细胞.

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