Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

A Publisher Correction to this article was published on 27 January 2020

This article has been updated

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Change history

  • 27 January 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 1.

    Farzadfard, F. & Lu, T. K. Emerging applications for DNA writers and molecular recorders. Science 361, 870–875 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Frieda, K. L. et al. Synthetic recording and in situ readout of lineage information in single cells. Nature 541, 107–111 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    McKenna, A. et al. Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 353, aaf7907 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Alemany, A., Florescu, M., Baron, C. S., Peterson-Maduro, J. & van Oudenaarden, A. Whole-organism clone tracing using single-cell sequencing. Nature 556, 108–112 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Kalhor, R et al. Developmental barcoding of whole mouse via homing CRISPR. Science 361, eaat9804 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Raj, B. et al. Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain. Nat. Biotechnol. 36, 442–450 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Spanjaard, B. et al. Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars. Nat. Biotechnol. 36, 469–473 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Farzadfard, F et al. Single-nucleotide-resolution computing and memory in living cells. Mol. Cell 75, 769–780 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Tang, W. & Liu, D. R. Rewritable multi-event analog recording in bacterial and mammalian cells. Science 360, eaap8992 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Kebschull, J. M. & Zador, A. M. Cellular barcoding: lineage tracing, screening and beyond. Nat. Methods 15, 871–879 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Kalhor, R., Mali, P. & Church, G. M. Rapidly evolving homing CRISPR barcodes. Nat. Methods 14, 195–200 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Pei, W. et al. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo. Nature 548, 456–460 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Shah, S., Lubeck, E., Zhou, W. & Cai, L. seqFISH accurately detects transcripts in single cells and reveals robust spatial organization in the hippocampus. Neuron 94, 752–758 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Shah, S. et al. Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH. Cell 174, 363–376 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Choi, H. M. T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Choi, H. M. T. et al. Third-generation hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145, dev165753 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Rouhanifard, S. H. et al. ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification. Nat. Biotechnol. 37, 84–89 (2018).

    Article  CAS  Google Scholar 

  23. 23.

    Marras, S. A. E., Bushkin, Y. & Tyagi, S. High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules. Proc. Natl Acad. Sci. USA 116, 13921–13926 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Mitra, R. D. et al. Fluorescent in situ sequencing on polymerase colonies. Anal. Biochem. 320, 55–65 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Lee, J. H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360–1363 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Chen, X., Sun, Y.-C., Church, G. M., Lee, J. H. & Zador, A. M. Efficient in situ barcode sequencing using padlock probe-based BaristaSeq. Nucleic Acids Res. 46, e22 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Feldman, D. et al. Optical pooled screens in human cells. Cell 179, 787–799 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Shah, S. et al. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing. Development 143, 2862–2867 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Sousa, R. & Mukherjee, S. T7 RNA polymerase. Prog. Nucleic Acid Res. Mol. Biol. 73, 1–41 (2003).

    Article  CAS  Google Scholar 

  31. 31.

    Choi, H. M. T. et al. Mapping a multiplexed zoo of mRNA expression. Development 143, 3632–3637 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Vieregg, J. R., Nelson, H. M., Stoltz, B. M. & Pierce, N. A. Selective nucleic acid capture with shielded covalent probes. J. Am. Chem. Soc. 135, 9691–9699 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Levesque, M. J., Ginart, P., Wei, Y. & Raj, A. Visualizing SNVs to quantify allele-specific expression in single cells. Nat. Methods 10, 865–867 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Sternberg, J. B. & Pierce, N. A. Exquisite sequence selectivity with small conditional RNAs. Nano Lett. 14, 4568–4572 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Wu, L. R. et al. Continuously tunable nucleic acid hybridization probes. Nat. Methods 12, 1191–1196 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Symmons, O. et al. Allele-specific RNA imaging shows that allelic imbalances can arise in tissues through transcriptional bursting. PLoS Genet. 15, e1007874 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    Article  CAS  Google Scholar 

  38. 38.

    Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: Abase editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Li, X. et al. Base editing with a Cpf1–cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Gehrke, J. M. et al. An APOBEC3A–Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Chan, M. et al. Molecular recording of mammalian embryogenesis. Nature 570, 77 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92 (1951).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Lois, C. & Alvarez-Buylla, A. Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145–1148 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Emanuel, G., Moffitt, J. R. & Zhuang, X. High-throughput, image-based screening of pooled genetic-variant libraries. Nat. Methods 14, 1159–1162 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Faedo, A. et al. Developmental expression of the T-box transcription factor T-bet/Tbx21 during mouse embryogenesis. Mech. Dev. 116, 157–160 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Baker, H., Kawano, T., Margolis, F. L. & Joh, T. H. Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat. J. Neurosci. 3, 69–78 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Lu, R., Neff, N. F., Quake, S. R. & Weissman, I. L. Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat. Biotechnol. 29, 928–933 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Naik, S. H. et al. Diverse and heritable lineage imprinting of early haematopoietic progenitors. Nature 496, 229–232 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Biddy, B et al. Single-cell mapping of lineage and identity in direct reprogramming. Nature 564, 219–224 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Weinreb, C., Rodriguez-Fraticelli, A. E., Camargo, F. D. & Klein, A. M. Lineage tracing on transcriptional landscapes links state to fate during differentiation. Preprint at https://www.biorxiv.org/content/10.1101/467886v2 (2018).

  53. 53.

    Walsh, C. & Cepko, C. L. Widespread dispersion of neuronal clones across functional regions of the cerebral cortex. Science 255, 434–440 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Kebschull, J. M. et al. High-throughput mapping of single-neuron projections by sequencing of barcoded RNA. Neuron 91, 975–987 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Bhang, H.-E. C. et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nat. Med. 21, 440–448 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Dixit, A. et al. Perturb-Seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Boutros, M., Heigwer, F. & Laufer, C. Microscopy-based high-content screening. Cell 163, 1314–1325 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Eng, C.-H. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature 568, 235–239 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Chen, R. et al. A barcoding strategy enabling higher-throughput library screening by microscopy. ACS Synth. Biol. 4, 1205–1216 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Weinstein, J. A., Regev, A. & Zhang, F. DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction. Cell 178, 229–241 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Listgarten, J. et al. Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs. Nat. Biomed. Eng. 2, 38–47 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    SantaLucia, J. Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl Acad. Sci. USA 95, 1460–1465 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Lois, C., Hong, E. J., Pease, S., Brown, E. J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013).

  68. 68.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Ding, F. & Elowitz, M. B. Constitutive splicing and economies of scale in gene expression. Nat. Struct. Mol. Biol. 26, 424–432 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Press, W. H. et al. Numerical Recipes in C: The Art of Scientific Computing (Cambridge Univ., 1992).

Download references

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

Affiliations

Authors

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.

Corresponding author

Correspondence to Michael B. Elowitz.

Ethics declarations

Competing interests

Authors have submitted a provisional patent application that is based on the technology described in this manuscript.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–18 and Supplementary Table 2

Reporting Summary

Supplementary Table 1

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/s41587-019-0299-4

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing