Abstract
The elimination of large genomic regions has been enabled by the advent of site-specific nucleases. However, as the intended deletions get larger, the efficiency of successful engineering decreases to a point where it is not feasible to retrieve edited cells due to the rarity of on-target events. To address this issue, we developed a system called molecular alteration of chromosomes with engineered tandem elements (MACHETE). MACHETE is a CRISPR–Cas9-based system involving two stages: the initial insertion of a bicistronic positive/negative selection cassette to the locus of interest. This is followed by the introduction of single-guide RNAs flanking the knockin cassette to engineer the intended deletion, where only cells that have lost the locus survive the negative selection. In contrast to other approaches optimizing the activity of sequence-specific nucleases, MACHETE selects for the deletion event itself, thus greatly enriching for cells with the engineered alteration. The procedure routinely takes 4–6 weeks from design to selection of polyclonal populations bearing the deletion of interest. We have successfully deployed MACHETE to engineer deletions of up to 45 Mb, as well as the rapid creation of allelic series to map the relevant activities within a locus. This protocol details the design and step-by-step procedure to engineer megabase-sized deletions in cells of interest, with potential application for cancer genetics, transcriptional regulation, genome architecture and beyond.
Key points
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MACHETE is a clustered regularly interspaced short palindromic repeats directed Cas9-based, two-stage system for creating megabase-sized genomic deletions: first, a bicistronic positive/negative selection cassette is inserted into the locus of interest, then single-guide RNAs are added to engineer the intended deletion. Only cells that have lost the locus survive the negative selection, thus greatly enriching for cells with deletions.
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MACHETE circumvents the inefficiency of retrieving cells with large deletions that limits previous methods.
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Data availability
All data related to this protocol are based on our previously published work14.
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Acknowledgements
We thank all members of the Lowe laboratory for advice and helpful discussions. F.M.B. was supported by a Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center (GMTEC) Postdoctoral Fellowship, a Memorial Sloan Kettering Cancer Center (MSKCC) Translational Research Oncology Training Fellowship (5T32CA160001-08) and a Young Investigator Award by the Edward P. Evans Foundation. This work was also supported by MSKCC’s David Rubenstein Center for Pancreatic Research Pilot Project (to S.W.L.), the Agilent Thought Leader Program (to S.W.L.) and the GMTEC Classic Individual Funding (to S.W.L.). S.W.L. is an investigator in the Howard Hughes Medical Institute and the Geoffrey Beene Chair for Cancer Biology. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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F.M.B. designed and developed the protocol with supervision from S.W.L. Both authors wrote and edited the manuscript.
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S.W.L. is a consultant and holds equity in Blueprint Medicines, ORIC Pharmaceuticals, Mirimus, PMV Pharmaceuticals, Faeth Therapeutics, and Senescea Therapeutics and is a consultant for Fate Therapeutics. F.M.B. declares no competing interests.
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Nature Protocols thanks Jiazhi Hu, Ophir Shalem and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Barriga, F. M. et al. Nat. Cancer (2022): https://doi.org/10.1038/s43018-022-00443-5
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Unprocessed gels
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Barriga, F.M., Lowe, S.W. Engineering megabase-sized genomic deletions with MACHETE (Molecular Alteration of Chromosomes with Engineered Tandem Elements). Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00953-9
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DOI: https://doi.org/10.1038/s41596-024-00953-9
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