Gene editing by engineered nucleases has revolutionized the field of gene therapy by enabling targeted and precise modification of the genome. However, the limited availability of methods for clonal tracking of edited cells has resulted in a paucity of information on the diversity, abundance and behavior of engineered clones. Here we detail the wet laboratory and bioinformatic BAR-Seq pipeline, a strategy for clonal tracking of cells harboring homology-directed targeted integration of a barcoding cassette. We present the BAR-Seq web application, an online, freely available and easy-to-use software that allows performing clonal tracking analyses on raw sequencing data without any computational resources or advanced bioinformatic skills. BAR-Seq can be applied to most editing strategies, and we describe its use to investigate the clonal dynamics of human edited hematopoietic stem/progenitor cells in xenotransplanted hosts. Notably, BAR-Seq may be applied in both basic and translational research contexts to investigate the biology of edited cells and stringently compare editing protocols at a clonal level. Our BAR-Seq pipeline allows library preparation and validation in a few days and clonal analyses of edited cell populations in 1 week.

工程核酸酶的基因编辑通过实现基因组的靶向和精确修饰，彻底改变了基因治疗领域。然而，用于编辑细胞克隆追踪的方法的可用性有限导致了关于工程克隆的多样性、丰度和行为的信息的匮乏。在这里，我们详细介绍了湿实验室和生物信息学 BAR-Seq 管道，这是一种克隆跟踪带有条形码盒同源定向靶向整合的细胞的策略。我们介绍了 BAR-Seq 网络应用程序，这是一种在线、免费且易于使用的软件，无需任何计算资源或高级生物信息学技能，即可对原始测序数据执行克隆跟踪分析。BAR-Seq 可应用于大多数编辑策略，我们描述了它在研究异种移植宿主中人类编辑的造血干细胞/祖细胞的克隆动力学方面的用途。值得注意的是，BAR-Seq 可应用于基础和转化研究环境，以研究编辑细胞的生物学并在克隆水平上严格比较编辑方案。我们的 BAR-Seq 流水线允许在几天内完成文库制备和验证，并在 1 周内对编辑后的细胞群进行克隆分析。