清脆的
诱导多能干细胞
生物
表型筛选
体细胞
表型
计算生物学
基因敲除
遗传筛选
Cas9
基因组编辑
药物发现
电池类型
遗传学
基因
细胞
生物信息学
胚胎干细胞
作者
Masataka Nishiga,Lei S. Qi,Joseph C. Wu
出处
期刊:Methods in molecular biology
日期:2021-01-01
卷期号:: 261-281
被引量:13
标识
DOI:10.1007/978-1-0716-1484-6_23
摘要
Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.
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