BAR-Seq clonal tracking of gene-edited cells

• Published in: Nature protocols Vol. 16; no. 6; pp. 2991 - 3025
• Main Authors: Ferrari, Samuele, Beretta, Stefano, Jacob, Aurelien, Cittaro, Davide, Albano, Luisa, Merelli, Ivan, Naldini, Luigi, Genovese, Pietro
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2021; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/794; 631/61/201/2110; Analytical Chemistry; Applications programs; Biological Techniques; Biomedical and Life Sciences; Cell Tracking - methods; Cells (biology); Clone Cells; Cloning; Computational Biology/Bioinformatics; Computer applications; DNA Barcoding, Taxonomic; Gene Editing; Gene therapy; Genetic modification; Genetic research; Genome editing; Genomes; Hematopoietic stem cells; Homology; Integration; Life Sciences; Methods; Microarrays; Next-generation sequencing; Nuclease; Organic Chemistry; Progenitor cells; Protocol; Software; Tracking; Web applications; Xenografts; Italy
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-021-00529-x

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. In this protocol, barcodes are introduced into cells via homology-directed targeted integration, and clones are tracked in xenotransplanted hosts by high-throughput sequencing. The results can be analyzed using a freely available online program.