利用 CRISPR 技术在人诱导多能干细胞中进行高效双等位基因标记。

Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR.

机构信息

Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA.

出版信息

STAR Protoc. 2023 Mar 17;4(1):102084. doi: 10.1016/j.xpro.2023.102084. Epub 2023 Feb 1.

DOI:10.1016/j.xpro.2023.102084

PMID:36853689

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9918420/

Abstract

Allelic tagging of endogenous genes enables studying gene function and transcriptional control in the native genomic context. Here, we present an efficient protocol for bi-allelic tagging of protein-coding genes with fluorescent reporters in human iPSCs using the CRISPR-Cas9-mediated homology-directed repair. We detail steps for design, cloning, electroporation, and single-cell clone isolation and validation. The tagging strategy described in this protocol is readily applicable for knockin of other reporters in diverse cell types for biomedical research.

摘要

等位基因标记内源性基因可在天然基因组环境中研究基因功能和转录调控。本研究使用 CRISPR-Cas9 介导的同源定向修复，在人诱导多能干细胞中建立了一种高效的双等位基因标记蛋白编码基因的荧光报告基因方法。文中详细介绍了设计、克隆、电穿孔、单细胞克隆分离和验证的步骤。该方法可用于多种细胞类型中其他报告基因的敲入，适用于生物医学研究。