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Rapidly evolving homing CRISPR barcodes

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

Abstract

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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Acknowledgements

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

Affiliations

  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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Contributions

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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DOI

https://doi.org/10.1038/nmeth.4108