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.
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Data availability
The BAR-Seq software with some example datasets is provided as a zip file (Supplementary Software) in the Supplementary Information. These datasets, which are part of the study originally described in ref. 9 and available in Gene Expression Omnibus with the accession code GSE144340, have been analyzed with the BAR-Seq computational pipeline to generate the example results presented in Fig. 4.
Code availability
The scripts for BAR-Seq analysis are freely available at https://bitbucket.org/bereste/bar-seq under the terms of the GNU General Public License version 3 (GPLv3). The BAR-Seq webtool is freely available at http://www.bioinfotiget.it/barseq.
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Acknowledgements
We thank all members of L.N.’s laboratory for discussion, the IRCCS San Raffaele Hospital Flow Cytometry facility (FRACTAL), the IRCCS San Raffaele Center for Omics Sciences (COSR), A. Auricchio and M. Doria (Telethon Institute of GEnetics and Medicine, the TIGEM Vector Core, Pozzuoli, Italy) for providing AAV6 vectors, L. Periè (Institute Curie, Paris, France) and J. Urbanus (the Netherlands Cancer Institute, Amsterdam, the Netherlands) for advice on the BAR cloning strategy, G. Schiroli (SR-Tiget) for initial help with the design of the BAR-Seq strategy, D. Lazarevic (COSR) for help with BAR-Seq amplicon sequencing and A. Calabria (SR-Tiget) for help with richness estimation. This work was supported by grants to: L.N. from Telethon (TIGET grant E4), the Italian Ministry of Health (PE-2016-02363691; E-Rare-3 JTC 2017), the Italian Ministry of University and Research (PRIN 2017 Prot. 20175XHBPN), the EU Horizon 2020 Program (UPGRADE) and the Louis-Jeantet Foundation through the 2019 Jeantet-Collen Prize for Translational Medicine; P.G. from Telethon (TIGET grant E3) and the Italian Ministry of Health (GR-2013-02358956). S.F. conducted this study as partial fulfillment of his Ph.D. in Molecular Medicine, International Ph.D. School, Vita-Salute San Raffaele University (Milan, Italy). A.J. conducted this study as partial fulfillment of his Ph.D. in Translational and Molecular Medicine (DIMET), Milano-Bicocca University (Monza, Italy).
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Contributions
S.F., P.G. and L.N conceived and developed the protocol. S.B., D.C. and I.M. developed the BAR-Seq bioinformatic pipeline. S.B. and I.M. developed the online BAR-Seq tool. S.F., A.J., L.A. and P.G. developed and optimized the gene-editing protocol. S.F., S.B., A.J., P.G., I.M. and L.N. wrote the manuscript. I.M., L.N. and P.G. supervised the work and share senior authorship.
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Competing interests
L.N. and P.G. are inventors of patents on applications of gene editing in HSPCs owned and managed by the San Raffaele Scientific Institute and the Telethon Foundation, including a patent application on improved gene editing filed by S.F., A.J., P.G. and L.N. L.N. is founder and quota holder and P.G. is quota holder of GeneSpire, a startup company aiming to develop ex vivo gene editing in genetic diseases. All other authors declare no competing interests.
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Peer review information Nature Protocols thanks Cynthia Dunbar, Diego A. Espinoza and Alejo E. Rodriguez-Fraticelli for their contribution to the peer review of this work.
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Related links
Key reference using this protocol
Ferrari, S. et al. Nat. Biotechnol. 38, 1298–1308 (2020): https://doi.org/10.1038/s41587-020-0551-y
Supplementary information
Supplementary Information
Supplementary Fig. 1.
Supplementary Software
BAR-Seq software and example datasets
Supplementary Tables 1–3
Tables of probability collisions and list of BAR-Seq primers
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Ferrari, S., Beretta, S., Jacob, A. et al. BAR-Seq clonal tracking of gene-edited cells. Nat Protoc 16, 2991–3025 (2021). https://doi.org/10.1038/s41596-021-00529-x
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DOI: https://doi.org/10.1038/s41596-021-00529-x
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