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Published January 2020 | Accepted Version + Supplemental Material
Journal Article Open

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription


Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

Additional Information

© 2019 Springer Nature Limited. Receive 18 December 2018; Revised 23 September 2019; Accepted 28 September 2019; Published 18 November 2019. Data availability: Data that are not included in the paper are available at https://data.caltech.edu/records/1303 (https://doi.org/10.22002/D1.1303) or from the corresponding author. Code availability: Scripts for all analyses presented in this paper are available at https://data.caltech.edu/records/1303 (https://doi.org/10.22002/D1.1303) or from the corresponding author. We are grateful to M. Schwartzkopf, H. Choi and N. Pierce for advice with HCR; K. Chow for help with cell culture; S. Shah for insightful discussions; and F. Ding for advice on image analysis. We also thank all the members of Elowitz, Cai and Lois laboratories for helpful discussions and critical feedback. Some of the imaging for this paper was performed in the Biological Imaging Facility with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. The research was funded by the National Institutes of Health (NIH) (grant R01 MH116508 to M.B.E., C.L. and L.C.), the Paul G. Allen Frontiers Group and Prime Awarding Agency (grant UWSC10142 to M.B.E., C.L. and L.C.), the Jane Coffin Childs Memorial Fund for Medical Research (grant 61-1650 to A.A.) and an NIH–NRSA training grant (T32 GM07616 to D.M.C.). M.B.E. is a Howard Hughes Medical Institute investigator. Author Contributions: A.A., L.S.-G., L.C., C.L. and M.B.E. designed research. A.A., L.S.-G., J.M.L. and M.W.B. performed experiments. A.A., D.M.C. and M.B.E. analyzed data. A.A. and M.B.E. wrote the manuscript. Competing interests: Authors have submitted a provisional patent application that is based on the technology described in this manuscript.


In the version of this article initially published, the y-axis label in Fig. 1g read "Proportion of cells with active site(s) (%)." The correct label is "Proportion of cells with no active site(s) (%)." And, near the bottom of Fig. 4a, the probe for original variant (orange) was shown with a C nucleotide and the probe for edited base variants (red) was shown with T; these nucleotides were switched with each other. The errors have been corrected in the HTML and PDF versions of the article.

Attached Files

Accepted Version - nihms-1540812.pdf

Supplemental Material - 41587_2019_299_MOESM1_ESM.pdf

Supplemental Material - 41587_2019_299_MOESM2_ESM.pdf

Supplemental Material - 41587_2019_299_MOESM3_ESM.xlsx


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August 19, 2023
October 20, 2023