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Rewritable digital data storage in live cells via engineered control of recombination directionality
Jerome Bonnet, Pakpoom Subsoontorn, and Drew Endy1
Department of Bioengineering, Room 269B, Y2E2 Building, 473 Via Ortega, Stanford University, Stanford, CA 94305 Edited by David Baker, University of Washington, Seattle, WA, and approved April 6, 2012 (received for review February 8, 2012)DNA inversion ∣ synthetic biology ∣ genetic engineering ∣ standard biological parts

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ost engineered genetic data storage systems use auto- or cross-regulating bistable systems of transcription repressors or activators to define and hold state via continuous gene expression (1–4). Such epigenetic storage systems can be subject to evolutionary counter selection due to resource burdens placed onthe host cell or spontaneous switching due to putatively stochastic fluctuations in cellular processes, including gene expression. Moreover, heterologous expression-based systems are difficult to redeploy given differences in gene regulatory mechanisms across organisms. Another approach for storing data inside organisms is to code extrinsic information within genetic material (5). Nucleic acidshave undergone natural selection to serve as heritable data storage material in organismal lineages. Moreover, DNA provides attractive features in terms of data storage robustness, scalability, and stability (6). In addition, engineered transmission of DNA molecules could support data exchange between organisms as needed to implement higher-order multicellular behaviors within programmed consortia(6, 7). Practically, researchers have begun to use enzymes that modify DNA, typically site-specific recombinases, to study and control engineered genetic systems. For example, recombinases can catalyze strand exchange between specific DNA sequences and enable precise manipulation of DNA in vitro and in vivo (8). Depending on the relative location or orientation of recombination sites, threedistinct recombination outcomes, integration, excision or inversion, can be realized. From such knowledge, several natural recombination systems have been reapplied to support research in cell and developmenwww.pnas.org/cgi/doi/10.1073/pnas.1202344109

Author contributions: J.B., P.S., and D.E. designed research; J.B. and P.S. performed research; J.B., P.S., and D.E. analyzed data; and J.B., P.S., andD.E. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option.
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To whom correspondence should be addressed. E-mail: endy@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1202344109/-/DCSupplemental.

PNAS Early Edition∣

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ENGINEERING

The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred celldivision or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionasefunctions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD...