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Rapidly evolving homing CRISPR barcodes

Nature Methods volume 14, pages 195200 (2017) | Download Citation


We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9–hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR–Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

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  1. 1.

    , , & The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119 (1983).

  2. 2.

    & Lineage tracing. Cell 148, 33–45 (2012).

  3. 3.

    , & Cell lineage analysis by intracellular injection of a tracer enzyme. Science 202, 1295–1298 (1978).

  4. 4.

    & Using Flp-recombinase to characterize expansion of Wnt1-expressing neural progenitors in the mouse. Dev. Biol. 201, 57–65 (1998).

  5. 5.

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

  6. 6.

    , , & Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo. Genome Biol. 15, R75 (2014).

  7. 7.

    , , & Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat. Biotechnol. 29, 928–933 (2011).

  8. 8.

    , & Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

  9. 9.

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

  10. 10.

    , & in The Future of the Brain: Essays by the World's Leading Neuroscientists (eds. Marcus, G. & Freeman, J.) 50–66 (Princeton University Press, 2016).

  11. 11.

    , & In vivo generation of DNA sequence diversity for cellular barcoding. Nucleic Acids Res. 42, e127 (2014).

  12. 12.

    , & Cellular barcoding: a technical appraisal. Exp. Hematol. 42, 598–608 (2014).

  13. 13.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  14. 14.

    et al. PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing. Nat. Commun. 6, 10124 (2015).

  15. 15.

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

  16. 16.

    et al. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat. Protoc. 10, 442–458 (2015).

  17. 17.

    et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

  18. 18.

    et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

  19. 19.

    & Emerging imaging and genomic tools for developmental systems biology. Dev. Cell 36, 597–610 (2016).

  20. 20.

    et al. Massively parallel whole-organism lineage tracing using CRISPR/Cas9 induced genetic scars. Preprint at bioRxiv (2016).

  21. 21.

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

  22. 22.

    , & Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science 353, aag0511 (2016).

  23. 23.

    et al. Physical principles for scalable neural recording. Front. Comput. Neurosci. 7, 137 (2013).

  24. 24.

    et al. Conneconomics: The Economics of Dense, Large-Scale, High-Resolution Neural Connectomics. Preprint at bioRxiv (2013).

  25. 25.

    et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

  26. 26.

    et al. Tracking genome engineering outcome at individual DNA breakpoints. Nat. Methods 8, 671–676 (2011).

  27. 27.

    , , , & Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

  28. 28.

    et al. Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 41, 9049–9061 (2013).

  29. 29.

    et al. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Res. 43, e37 (2015).

  30. 30.

    , & The New S Language: A Programming Environment for Data Analysis and Graphics (Chapman & Hall/CRC, 1988).

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The authors would like to acknowledge W.L. Chew, J. Aach, S. Byrne, E. Daugharthy, T. Ferrante, J.H. Lee, M. Moosburner, I. Peikon, H. Lee, A. Ng, J. Fernandez Juarez, A. Marblestone, A. Chavez, Y. Mayshar, J. Scheiman, K. Kalhor, T. Wu, J. Shendure, and T. Lu for helpful comments or discussions and the Biopolymers Facility at HMS for technical assistance. This work has been supported by funding from NIH grants MH103910 and HG005550 (G.M.C.) and the Intelligence Advanced Research Projects Activity (IARPA) via Department of Interior/Interior Business Center (DoI/IBC) contract number D16PC00008 (G.M.C.), and by UCSD new faculty startup funds (P.M.).

Author information


  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Reza Kalhor
    •  & George M Church
  2. Department of Bioengineering, University of California San Diego, La Jolla, California, USA.

    • Prashant Mali
  3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.

    • George M Church


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R.K., P.M., and G.M.C. conceived the study. R.K. and P.M. carried out the experiments. R.K. analyzed the data. R.K. and P.M. wrote the manuscript.

Competing interests

R.K., P.M. and G.M.C. have filed provisional patent applications based on this study.

Corresponding authors

Correspondence to Prashant Mali or George M Church.

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