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.

Ferrari, S., Beretta, S., Jacob, A., Cittaro, D., Albano, L., Merelli, I., et al. (2021). BAR-Seq clonal tracking of gene-edited cells. NATURE PROTOCOLS, 16(6), 2991-3025 [10.1038/s41596-021-00529-x].

BAR-Seq clonal tracking of gene-edited cells

Beretta S.;Jacob A.;Merelli I.;
2021

Abstract

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.
Articolo in rivista - Articolo scientifico
Cell Tracking; DNA Barcoding, Taxonomic; Clone Cells; Gene Editing; Software
English
24-mag-2021
2021
16
6
2991
3025
none
Ferrari, S., Beretta, S., Jacob, A., Cittaro, D., Albano, L., Merelli, I., et al. (2021). BAR-Seq clonal tracking of gene-edited cells. NATURE PROTOCOLS, 16(6), 2991-3025 [10.1038/s41596-021-00529-x].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/324821
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