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.

中文翻译：

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使用独立于同源性的CRISPR-Cas9精确基因组编辑快速高效地产生敲入式人类器官。

CRISPR-Cas9技术彻底改变了基因组编辑，适用于类器官领域。然而，缺乏将外源DNA序列精确整合到人类器官中的稳健的敲入方法。在这里，我们描述了CRISPR-Cas9介导的不依赖同源性的类器官转基因（CRISPR-HOT），它能够有效产生代表不同组织的敲入人类器官。CRISPR-HOT避免了广泛的克隆，并且在实现将外源DNA序列精确整合到所需基因座中的过程中胜过了同源指导修复（HDR），而无需灭活未转化细胞中的TP53，后者以前曾被用来增加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，并生成罕见肠道细胞类型的报告基因系。双重报告基因，其中有丝分裂纺锤体被内源性标记的微管蛋白标记，细胞膜被内源性标记的E-钙粘着蛋白揭露的人类肝细胞分裂模式。结合微管蛋白标签和TP53敲除表明TP53参与控制肝细胞倍性和有丝分裂纺锤体保真度。CRISPR-HOT简化了人类类器官的基因组编辑。