Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing
Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing
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DOI：
10.1038/s41556-020-0472-5
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发表时间：
2020-03-02
影响因子：
21.3
通讯作者：
Clevers, Hans
中科院分区：
文献类型：
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作者：
Artegiani, Benedetta;Hendriks, Delilah;Clevers, Hans
CRISPR-Cas9技术彻底改变了基因组编辑，适用于类器官领域。然而，将外源DNA序列精确整合到人类类器官中缺乏稳健的敲入方法。在这里，我们描述了CRISPR-Cas9介导的同源非依赖性类器官转基因（CRISPR-HOT），它能够高效生成代表不同组织的敲入人类类器官。CRISPR-HOT避免了广泛的克隆，并且在实现外源DNA序列精确整合到所需基因座中方面优于同源定向修复（HDR），而不需要在未转化的细胞中使TP 53失活，这之前用于增加HDR介导的敲入。CRISPR-HOT用于荧光标记和可视化亚细胞结构分子，并生成罕见肠细胞类型的报告细胞系。一个双标记，其中有丝分裂纺锤体标记的内源性标记的微管蛋白和细胞膜的内源性标记的E-钙粘蛋白-人类肝细胞分裂的模式。结合微管蛋白标记与TP 53敲除揭示TP 53参与控制肝细胞倍性和有丝分裂纺锤体保真度。Artegiani，Hendriks等人描述了一种基于CRISPR-Cas9的方法，该方法使用非同源末端连接来研究罕见的肠细胞类型和人类肝细胞分裂，从而有效地产生人类敲入类器官。
CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.Artegiani, Hendriks et al. describe a CRISPR-Cas9-based method to efficiently generate human knock-in organoids using non-homologous end joining to study rare intestinal cell types and human hepatocyte division.