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

Abstract

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.

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Fig. 1: Phage RNA polymerases enable in situ readout of DNA barcodes without in vivo expression.
Fig. 2: Reliable detection of short barcodes.
Fig. 3: Probe competition accurately discriminates SNVs.
Fig. 4: CRISPR base edits can be read out in situ.
Fig. 5: Zombie can detect barcodes and discriminate SNVs in chick embryo and adult mouse brain.
Fig. 6: In situ readout of a combinatorial barcode library.

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.

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Acknowledgements

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 information

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.

Correspondence to Michael B. Elowitz.

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Supplementary Information

Supplementary Figs. 1–18 and Supplementary Table 2

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Supplementary Table 1

Sequences of all the new constructs, barcodes and probes used in the study organized by the corresponding figures.

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Askary, A., Sanchez-Guardado, L., Linton, J.M. et al. In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription. Nat Biotechnol 38, 66–75 (2020) doi:10.1038/s41587-019-0299-4

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