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Temporally resolved transcriptional recording in E. coli DNA using a Retro-Cascorder


Biological signals occur over time in living cells. Yet most current approaches to interrogate biology, particularly gene expression, use destructive techniques that quantify signals only at a single point in time. A recent technological advance, termed the Retro-Cascorder, overcomes this limitation by molecularly logging a record of gene expression events in a temporally organized genomic ledger. The Retro-Cascorder works by converting a transcriptional event into a DNA barcode using a retron reverse transcriptase and then storing that event in a unidirectionally expanding clustered regularly interspaced short palindromic repeats (CRISPR) array via acquisition by CRISPR–Cas integrases. This CRISPR array-based ledger of gene expression can be retrieved at a later point in time by sequencing. Here we describe an implementation of the Retro-Cascorder in which the relative timing of transcriptional events from multiple promoters of interest is recorded chronologically in Escherichia coli populations over multiple days. We detail the molecular components required for this technology, provide a step-by-step guide to generate the recording and retrieve the data by Illumina sequencing, and give instructions for how to use custom software to infer the relative transcriptional timing from the sequencing data. The example recording is generated in 2 d, preparation of sequencing libraries and sequencing can be accomplished in 2–3 d, and analysis of data takes up to several hours. This protocol can be implemented by someone familiar with basic bacterial culture, molecular biology and bioinformatics. Analysis can be minimally run on a personal computer.

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Fig. 1: Plots summarizing the effect of the number of simulated informative arrays on ordering score accuracy.
Fig. 2: Retro-Cascorder experimental and computational workflow.
Fig. 3: Simulated ordering score results from different transcriptional programs.
Fig. 4: Illustrative ordering analysis of a recording experiment.

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Data availability

Sequencing data associated with this study are available in the NCBI SRA (PRJNA838025).

Code availability

The latest version of the analysis code can be accessed through our lab GitHub ( The release at time of manuscript publishing is available at


  1. Siuti, P., Yazbek, J. & Lu, T. K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P. & Endy, D. Amplifying genetic logic gates. Science 340, 599–603 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Yang, L. et al. Permanent genetic memory with >1-byte capacity. Nat. Methods 11, 1261–1266 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Farzadfard, F. & Lu, T. K. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Courbet, A., Endy, D., Renard, E., Molina, F. & Bonnet, J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci. Transl. Med. 7, 289ra83–289ra83 (2015).

    Article  PubMed  Google Scholar 

  6. Roquet, N., Soleimany, A. P., Ferris, A. C., Aaronson, S. & Lu, T. K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016).

    Article  PubMed  Google Scholar 

  7. Hsiao, V., Hori, Y., Rothermund, P. W. & Murray, M. M. A population-based temporal logic gate for timing and recording chemical events. Mol. Syst. Biol. 12, 869 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Weinberg, B. H. et al. Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat. Biotechnol. 35, 453–462 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Perli, S. D., Cui, C. H. & Lu, T. K. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science 353, aag0511 (2016).

    Article  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  11. Kempton, H. R., Love, K. S., Guo, L. Y. & Qi, L. S. Scalable biological signal recording in mammalian cells using Cas12a base editors. Nat. Chem. Biol. 1–9 (2022)

  12. Chen, W. et al. Multiplex genomic recording of enhancer and signal transduction activity in mammalian cells. Preprint at bioRxiv (2021).

  13. Loveless, T. B. et al. Molecular recording of sequential cellular events into DNA. Preprint at bioRxiv (2021).

  14. Choi, J. et al. A time-resolved, multi-symbol molecular recorder via sequential genome editing. Nature 608, 98–107 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. Molecular recordings by directed CRISPR spacer acquisition. Science 353, aaf1175 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Sheth, R. U., Yim, S. S., Wu, F. L. & Wang, H. H. Multiplex recording of cellular events over time on CRISPR biological tape. Science 358, 1457–1461 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Schmidt, F., Cherepkova, M. Y. & Platt, R. J. Transcriptional recording by CRISPR spacer acquisition from RNA. Nature 562, 380–385 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Yim, S. S. et al. Robust direct digital-to-biological data storage in living cells. Nat. Chem. Biol. 17, 246–253 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sheth, R. U. & Wang, H. H. DNA-based memory devices for recording cellular events. Nat. Rev. Genet. 19, 718–732 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lear, S. K. & Shipman, S. L. Molecular recording: transcriptional data collection into the genome. Curr. Opin. Biotechnol. 79, 102855 (2023).

    Article  CAS  PubMed  Google Scholar 

  21. Bhattarai-Kline, S. et al. Recording gene expression order in DNA by CRISPR addition of retron barcodes. Nature (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nuñez, J. K. et al. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nat. Struct. Mol. Biol. 21, 528–534 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547, 345–349 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Silas, S. et al. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase–Cas1 fusion protein. Science 351, aad4234 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Tanna, T., Schmidt, F., Cherepkova, M. Y., Okoniewski, M. & Platt, R. J. Recording transcriptional histories using Record-seq. Nat. Protoc. 15, 513–539 (2020).

    Article  CAS  PubMed  Google Scholar 

  27. Schmidt, F. et al. Noninvasive assessment of gut function using transcriptional recording sentinel cells. Science 376, eabm6038 (2022).

    Article  CAS  PubMed  Google Scholar 

  28. Yehl, K. & Lu, T. Scaling computation and memory in living cells. Curr. Opin. Biomed. Eng. 4, 143–151 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Nuñez, J. K., Bai, L., Harrington, L. B., Hinder, T. L. & Doudna, J. A. CRISPR immunological memory requires a host factor for specificity. Mol. Cell 62, 824–833 (2016).

    Article  PubMed  Google Scholar 

  30. Yoganand, K. N. R., Sivathanu, R., Nimkar, S. & Anand, B. Asymmetric positioning of Cas1–2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system. Nucleic Acids Res. 45, 367–381 (2017).

    Article  CAS  PubMed  Google Scholar 

  31. Sharon, E. et al. Functional genetic variants revealed by massively parallel precise genome editing. Cell 175, 544–557.e16 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kong, X. et al. Precise genome editing without exogenous donor DNA via retron editing system in human cells. Protein Cell 12, 899–902 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Lopez, S. C., Crawford, K. D., Lear, S. K., Bhattarai-Kline, S. & Shipman, S. L. Precise genome editing across kingdoms of life using retron-derived DNA. Nat. Chem. Biol. 18, 199–206 (2022).

    Article  CAS  PubMed  Google Scholar 

  34. Zhao, B., Chen, S.-A. A., Lee, J. & Fraser, H. B. Bacterial retrons enable precise gene editing in human cells. CRISPR J. 5, 31–39 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Palka, C., Fishman, C. B., Bhattarai-Kline, S., Myers, S. A. & Shipman, S. L. Retron reverse transcriptase termination and phage defense are dependent on host RNase H1. Nucleic Acids Res. 50, 3490–3504 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Munck, C., Sheth, R. U., Freedberg, D. E. & Wang, H. H. Recording mobile DNA in the gut microbiota using an Escherichia coli CRISPR–Cas spacer acquisition platform. Nat. Commun. 11, 95 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lee, P. Y., Costumbrado, J., Hsu, C. Y. & Kim, Y. H. Agarose gel electrophoresis for the separation of DNA fragments. J. Vis. Exp. 62, 3923 (2012).

    Google Scholar 

  38. Kluyver, T., et al. Jupyter Notebooks—a publishing format for reproducible computational workflows. In: Loizides, F. & Schmidt, B. (eds.) Positioning and Power in Academic Publishing: Players, Agents and Agendas, 87–90 (IOS Press, 2016).

  39. Joshi, N.A. & Fass, J.N. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (Version 1.33) Available at: (2011).

  40. JoVE Science Education Database. Microbiology. Serial Dilutions and Plating: Microbial Enumeration (JoVE, 2022).

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This work was supported by funding from the National Science Foundation (2137692), the NIH/NIGMS (1DP2GM140917-01) and the Pew Biomedical Scholars Program. S.L.S. is a Chan Zuckerberg Biohub investigator and acknowledges additional funding support from the L.K. Whittier Foundation. S.K.L. was supported by an NSF Graduate Research Fellowship (2034836). S.C.L. was supported by a Berkeley Fellowship for Graduate Study.

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Authors and Affiliations



S.K.L. and S.C.L. wrote the protocol, with S.K.L. focusing on the experimental components and S.C.L. focusing on the computational components. The protocol was revised on the basis of input from A.G.-D., S.B.-K. and S.L.S. Additional simulation data was generated and analyzed by S.K.L. and S.C.L. using the Spacer-Seq code adapted into a JupyterLab notebook written by S.C.L. Figures were contributed by S.K.L., S.C.L. and A.G.D.

Corresponding author

Correspondence to Seth L. Shipman.

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Competing interests

S.L.S. is a named inventor on a patent application assigned to Harvard College, ‘Method of recording multiplexed biological information into a CRISPR array using a retron’ (US20200115706A1). The remaining authors declare no competing interests.

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Nature Protocols thanks Michelle Chan 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

Bhattarai-Kline, S. et al. Nature 608, 217–225 (2022):

Supplementary information

Supplementary Information

Supplementary Method and Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Indexing primer sequences for multiplexing samples during library preparation.

Supplementary Table 2

KAPA Library Quantification Data Analysis Template as described for the KAPA Library Quantification Kit. Worksheet provides a readme page, an analysis page for data input, and a summary page for data output.

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Lear, S.K., Lopez, S.C., González-Delgado, A. et al. Temporally resolved transcriptional recording in E. coli DNA using a Retro-Cascorder. Nat Protoc 18, 1866–1892 (2023).

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