同基因疾病模型，例如基因工程类器官，可以深入了解遗传变异对器官功能的影响。在这里，我们提出了一个使用下一代 CRISPR 工具从成体干细胞衍生的类器官创建同基因疾病模型的方案。我们描述了单引导 RNA (sgRNA) 设计和克隆、电穿孔以及​​选择电穿孔细胞的步骤。然后我们详细介绍克隆系生成的程序。下一代 CRISPR 工具不需要双链断裂 (DSB) 诱导来发挥其功能，从而简化了体外疾病模型的生成。有关该协议的使用和执行的完整详细信息，请参阅 Geurts 等人 1,2。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.1,2.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.