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.
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Farzadfard, F. & Lu, T. K. Emerging applications for DNA writers and molecular recorders. Science 361, 870–875 (2018).
Frieda, K. L. et al. Synthetic recording and in situ readout of lineage information in single cells. Nature 541, 107–111 (2017).
McKenna, A. et al. Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 353, aaf7907 (2016).
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).
Kalhor, R et al. Developmental barcoding of whole mouse via homing CRISPR. Science 361, eaat9804 (2018).
Raj, B. et al. Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain. Nat. Biotechnol. 36, 442–450 (2018).
Spanjaard, B. et al. Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars. Nat. Biotechnol. 36, 469–473 (2018).
Farzadfard, F et al. Single-nucleotide-resolution computing and memory in living cells. Mol. Cell 75, 769–780 (2019).
Tang, W. & Liu, D. R. Rewritable multi-event analog recording in bacterial and mammalian cells. Science 360, eaap8992 (2018).
Kebschull, J. M. & Zador, A. M. Cellular barcoding: lineage tracing, screening and beyond. Nat. Methods 15, 871–879 (2018).
Kalhor, R., Mali, P. & Church, G. M. Rapidly evolving homing CRISPR barcodes. Nat. Methods 14, 195–200 (2017).
Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).
Pei, W. et al. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo. Nature 548, 456–460 (2017).
Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).
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).
Shah, S. et al. Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH. Cell 174, 363–376 (2018).
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).
Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).
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).
Choi, H. M. T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).
Choi, H. M. T. et al. Third-generation hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145, dev165753 (2018).
Rouhanifard, S. H. et al. ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification. Nat. Biotechnol. 37, 84–89 (2018).
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).
Mitra, R. D. et al. Fluorescent in situ sequencing on polymerase colonies. Anal. Biochem. 320, 55–65 (2003).
Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).
Lee, J. H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360–1363 (2014).
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).
Feldman, D. et al. Optical pooled screens in human cells. Cell 179, 787–799 (2019).
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).
Sousa, R. & Mukherjee, S. T7 RNA polymerase. Prog. Nucleic Acid Res. Mol. Biol. 73, 1–41 (2003).
Choi, H. M. T. et al. Mapping a multiplexed zoo of mRNA expression. Development 143, 3632–3637 (2016).
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).
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).
Sternberg, J. B. & Pierce, N. A. Exquisite sequence selectivity with small conditional RNAs. Nano Lett. 14, 4568–4572 (2014).
Wu, L. R. et al. Continuously tunable nucleic acid hybridization probes. Nat. Methods 12, 1191–1196 (2015).
Symmons, O. et al. Allele-specific RNA imaging shows that allelic imbalances can arise in tissues through transcriptional bursting. PLoS Genet. 15, e1007874 (2019).
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).
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).
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).
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).
Li, X. et al. Base editing with a Cpf1–cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).
Gehrke, J. M. et al. An APOBEC3A–Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).
Chan, M. et al. Molecular recording of mammalian embryogenesis. Nature 570, 77 (2019).
Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92 (1951).
Lois, C. & Alvarez-Buylla, A. Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145–1148 (1994).
Emanuel, G., Moffitt, J. R. & Zhuang, X. High-throughput, image-based screening of pooled genetic-variant libraries. Nat. Methods 14, 1159–1162 (2017).
Faedo, A. et al. Developmental expression of the T-box transcription factor T-bet/Tbx21 during mouse embryogenesis. Mech. Dev. 116, 157–160 (2002).
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).
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).
Naik, S. H. et al. Diverse and heritable lineage imprinting of early haematopoietic progenitors. Nature 496, 229–232 (2013).
Biddy, B et al. Single-cell mapping of lineage and identity in direct reprogramming. Nature 564, 219–224 (2018).
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).
Walsh, C. & Cepko, C. L. Widespread dispersion of neuronal clones across functional regions of the cerebral cortex. Science 255, 434–440 (1992).
Kebschull, J. M. et al. High-throughput mapping of single-neuron projections by sequencing of barcoded RNA. Neuron 91, 975–987 (2016).
Bhang, H.-E. C. et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nat. Med. 21, 440–448 (2015).
Dixit, A. et al. Perturb-Seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866 (2016).
Boutros, M., Heigwer, F. & Laufer, C. Microscopy-based high-content screening. Cell 163, 1314–1325 (2015).
Eng, C.-H. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature 568, 235–239 (2019).
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).
Chen, R. et al. A barcoding strategy enabling higher-throughput library screening by microscopy. ACS Synth. Biol. 4, 1205–1216 (2015).
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).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
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).
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).
SantaLucia, J. Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl Acad. Sci. USA 95, 1460–1465 (1998).
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).
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Ding, F. & Elowitz, M. B. Constitutive splicing and economies of scale in gene expression. Nat. Struct. Mol. Biol. 26, 424–432 (2019).
Press, W. H. et al. Numerical Recipes in C: The Art of Scientific Computing (Cambridge Univ., 1992).
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.
Authors have submitted a provisional patent application that is based on the technology described in this manuscript.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
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) doi:10.1038/s41587-019-0299-4
Current Opinion in Biotechnology (2020)