Abstract
Recording molecular information to genomic DNA is a powerful means of investigating topics ranging from multicellular development to cancer evolution. With molecular recording based on genome editing, events such as cell divisions and signaling pathway activity drive specific alterations in a cell’s DNA, marking the genome with information about a cell’s history that can be read out after the fact. Although genome editing has been used for molecular recording, capturing the temporal relationships among recorded events in mammalian cells remains challenging. The DNA Typewriter system overcomes this limitation by leveraging prime editing to facilitate sequential insertions to an engineered genomic region. DNA Typewriter includes three distinct components: DNA Tape as the ‘substrate’ to which edits accrue in an ordered manner, the prime editor enzyme, and prime editing guide RNAs, which program insertional edits to DNA Tape. In this protocol, we describe general design considerations for DNA Typewriter, step-by-step instructions on how to perform recording experiments by using DNA Typewriter in HEK293T cells, and example scripts for analyzing DNA Typewriter data (https://doi.org/10.6084/m9.figshare.22728758). This protocol covers two main applications of DNA Typewriter: recording sequential transfection events with programmed barcode insertions by using prime editing and recording lineage information during the expansion of a single cell to many. Compared with other methods that are compatible with mammalian cells, DNA Typewriter enables the recording of temporal information with higher recording capacities and can be completed within 4–6 weeks with basic expertise in molecular cloning, mammalian cell culturing and DNA sequencing data analysis.
Key points
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DNA Typewriter is a CRISPR genome editing-based method for recording the temporal order of molecular events by tracing the physical order of unique barcodes along a DNA Tape array.
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Compared with other existing methods, DNA Typewriter is highly multiplexable, unidirectional and sequential, capturing thousands of insertions in the precise order in which they occur, and it is active in living mammalian cells, including HEK293T, mouse embryonic stem cells and fibroblasts.
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Data availability
Example data files have been deposited to Figshare (https://doi.org/10.6084/m9.figshare.22728758.v1)37. Plasmids to clone in pegRNAs and DNA Tape have been deposited to Addgene (cat. nos. 200029 for lentiviral backbone construct and 200030 for piggyBAC backbone construct).
Code availability
Along with the example data, example scripts that can be directly used to analyze example data files have been deposited to Figshare (https://doi.org/10.6084/m9.figshare.22728758.v1)37. For recording the order of transfection, we have included an example script (TAPE_text_sorting.ipynb) to determine the order of transfected barcodes from paired-end sequencing data. For generating a single-cell lineage tree, we run a first custom script (TAPE_10X_read2fromBAM.ipynb) to extract the single-cell barcode information along with edits in captured DNA Tape molecules. The output can then be used in the second custom script (DNATypewriter_SingleCellLineage_Rscript.ipynb) to plot the lineage tree on the basis of the shared patterns of edited DNA Tapes from single cells.
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Acknowledgements
We thank R. Daza, B. Martin, H. Kim and J. Nathans for feedback on this manuscript and protocol. We thank members of Jay Shendure’s laboratory for developing the DNA Typewriter platform as well as David Liu’s laboratory for developing and improving the prime-editing technology. This work is supported by a grant from the Paul G. Allen Frontiers Group (Allen Discovery Center for Cell Lineage Tracing to J.S.) and the National Human Genome Research Institute (UM1HG011586 to J.S. and K99HG012973 to J.C.). H.L. is supported by the NSF Graduate Research Fellowship Program (DGE-2140004). J.C. was a Howard Hughes Medical Institute Fellow of the Damon Runyon Cancer Research Foundation (DRG-2403-20). J.S. is an investigator of the Howard Hughes Medical Institute.
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This protocol is based on a paper by J.C. and J.S. H.L. and J.C. contributed equally and wrote the manuscript and prepared the figures. J.S. supervised the research and wrote parts of the manuscript. All authors edited the manuscript.
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The University of Washington has filed a patent application partially based on this work, in which J.C. and J.S. are listed as inventors. J.S. is on the scientific advisory board, a consultant, and/or a co-founder of Prime Medicine, Cajal Neuroscience, Guardant Health, Maze Therapeutics, Camp4 Therapeutics, Phase Genomics, Adaptive Biotechnologies, Scale Biosciences, Sixth Street Capital and Pacific Biosciences. H.L. declares no competing interests.
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Nature Protocols thanks Nozomu Yachie 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
Choi, J. et al. Nature 608, 98–107 (2022): https://doi.org/10.1038/s41586-022-04922-8
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Supplementary Table 1
Supplementary Table 1
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Liao, H., Choi, J. & Shendure, J. Molecular recording using DNA Typewriter. Nat Protoc 19, 2833–2862 (2024). https://doi.org/10.1038/s41596-024-01003-0
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DOI: https://doi.org/10.1038/s41596-024-01003-0