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<jats:sec> <jats:title>INTRODUCTION</jats:title> <jats:p>Although recent advances in DNA synthesis and sequencing technologies have made practical the writing and readout of arbitrary data in the form of synthetic DNA, still lacking are the robust tools necessary to generate a dynamic record of such information within the genomes of living cells. An in vivo system, built out of biological parts with large storage capacity, would enable the recording of defined biological events into stable genetic memory and facilitate the tracking of long molecular and cellular histories.</jats:p> </jats:sec> <jats:sec> <jats:title>RATIONALE</jats:title> <jats:p>The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas system is a prokaryotic type of immunological memory. Foreign DNA sequences originating from viral infections are stored within genome-based arrays in the form of short sequences—called spacers—that confer sequence-specific resistance to the invading nucleic acids. These arrays not only preserve the spacer sequences but also record the order in which the sequences are acquired, generating a temporal record of acquisition events. We harnessed this system to record arbitrary DNA sequences into a genomic CRISPR array in the form of spacers acquired from synthetic oligonucleotides electroporated into a population of cells overexpressing the CRISPR adaptation proteins Cas1 and Cas2. This enabled the recording of defined molecular events into a stable genomic locus over time and the storage of arbitrary information across a population of cells.</jats:p> </jats:sec> <jats:sec> <jats:title>RESULTS</jats:title> <jats:p> We show that the Cas1-Cas2 complex can be used in vivo to integrate synthetic DNA of a defined sequence into the <jats:italic>Escherichia coli</jats:italic> genome. We used this feature to examine the type I-E CRISPR-Cas spacer acquisition process and optimized the synthetic spacer design to achieve higher acquisition efficiency and specific integration orientation through the addition of an AAG protospacer adjacent motif (PAM). We then generated stable genomic recordings of multiple molecular events by electroporating sets of oligonucleotides over several days. These molecular records were read out with high-throughput sequencing and then decoded with a program that identified and faithfully reconstructed the temporal event order. </jats:p> <jats:p> Last, we used directed evolution to generate many Cas1-Cas2 mutants with modified PAM specificity (PAM <jats:sup>NC</jats:sup> ). By modulating expression of these mutant and wild-type Cas1-Cas2 complexes, we could dynamically control the orientation of spacer integration. This enabled us to record acquisition events in multiple modes. That is, information was encoded in both the temporal order of the spacers and the orientation in which they were integrated. </jats:p> </jats:sec> <jats:sec> <jats:title>CONCLUSION</jats:title> <jats:p>Our results establish a recording system that uses the nucleotide content, temporal ordering, and orientation of defined DNA sequences within a CRISPR array in order to encode arbitrary information within the genomes of a population of cells. Because information can be encoded in spacer nucleotide space (up to two bits per base) and in alternate modes, the system has the potential to record and permanently store higher capacities of information than any other synthetic biological system to date. This lays the foundation for an in vivo recording device that could be coupled with diverse molecular phenomena and used for applications that require tracing of long molecular histories. We also demonstrate that delivery of synthetic DNA substrates to a CRISPR-Cas adaptation system in vivo is a practical method to probe and adapt the system.</jats:p> <jats:fig fig-type="figure" orientation="portrait" position="float"> <jats:caption> <jats:title>Two modes of encoding information into the CRISPR locus.</jats:title> <jats:p> ( <jats:bold>A</jats:bold> ) Oligonucleotides containing an AAG PAM and 32 variable bases were electroporated into cells overexpressing Cas1-Cas2 and inserted into the genomic CRISPR array. Delivery of oligos with distinct sequence over time generates a molecular record. ( <jats:bold>B</jats:bold> ) Cas1-Cas2 mutants identified through directed evolution alter the orientation of acquisition. Varying expression ratios of wild-type and mutant Cas1-Cas2 over time generates a record encoded in spacer orientation. </jats:p> </jats:caption> <jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" orientation="portrait" position="float" xlink:href="353_aaf1175_fa.jpeg" /> </jats:fig> </jats:sec>

<jats:sec> <jats:title>INTRODUCTION</jats:title> <jats:p> The developmental path by which a fertilized egg gives rise to the cells of a multicellular organism is termed the cell lineage. In 1983, John Sulston and colleagues documented the invariant cell lineage of the roundworm <jats:italic>Caenorhabditis elegans</jats:italic> as determined by visual observation. However, tracing cell lineage in nearly all other multicellular organisms is vastly more challenging. Contemporary methods rely on genetic markers or somatic mutations, but these approaches have limitations that preclude their application at the level of a whole, complex organism. </jats:p> </jats:sec> <jats:sec> <jats:title>RATIONALE</jats:title> <jats:p>For a technology to comprehensively trace cell lineages in a complex multicellular system, it must uniquely and incrementally mark cells and their descendants over many divisions and in a way that does not interfere with normal development. These unique marks must also accumulate irreversibly over time, allowing the reconstruction of lineage trees. Finally, the full set of marks must be read out from each of many single cells. We hypothesized that genome editing, which introduces diverse, irreversible edits in a highly programmable fashion, could be repurposed for cell lineage tracing in a way that realizes these characteristics.</jats:p> <jats:p>To this end, we developed a method termed genome editing of synthetic target arrays for lineage tracing (GESTALT). This method uses genome editing to generate a combinatorial diversity of mutations that accumulate over many cell divisions within a compact DNA barcode consisting of multiple clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 target sites. Lineage relationships can be readily queried by sequencing the edited barcodes and relating the patterns of edits observed.</jats:p> </jats:sec> <jats:sec> <jats:title>RESULTS</jats:title> <jats:p>We first developed this approach in cell culture, editing synthetic arrays of 9 to 12 CRISPR/Cas9 target sites to generate thousands of unique derivative barcodes. We show that edited barcodes can be read by targeted sequencing of either DNA or RNA. In addition, the rates and patterns of barcode editing are tunable and the diverse edits accumulate over successive divisions in a way that is informative of cell lineage.</jats:p> <jats:p> We then applied GESTALT to the zebrafish <jats:italic>Danio rerio</jats:italic> by injecting fertilized eggs with editing reagents that target a genomic barcode bearing 10 target sites. Across dozens of embryos, we demonstrate the accumulation of hundreds to thousands of uniquely edited barcodes per animal, from which lineage relationships can be inferred on the basis of shared mutations. In adult zebrafish, we evaluated the edited barcodes from ~200,000 cells and observed that the majority of cells in each organ are derived from a small number of progenitor cells. Furthermore, ancestral progenitors, inferred on the basis of shared mutations among subsets of cells, can contribute to different germ layers and organ systems. </jats:p> </jats:sec> <jats:sec> <jats:title>CONCLUSION</jats:title> <jats:p>Our proof-of-principle experiments show that combinatorial, cumulative genome editing of a compact barcode can be used to record lineage information in multicellular systems. Further optimization of GESTALT will enable mapping of the complete cell lineage in diverse organisms. This method could also be adapted to link cell lineage information to molecular profiles of the same cells. In the long term, we envision that rich, systematically generated maps of organismal development—wherein lineage, epigenetic, transcriptional, and positional information are concurrently captured at single-cell resolution—will advance our understanding of development in both healthy and disease states. More broadly, cumulative and combinatorial genome editing could stably record other types of biological information and history in living cells.</jats:p> <jats:fig fig-type="figure" orientation="portrait" position="float"> <jats:caption> <jats:title>GESTALT.</jats:title> <jats:p> ( <jats:bold>Left</jats:bold> ) A barcode of CRISPR/Cas9 target sites is progressively edited over many cell divisions. ( <jats:bold>Right</jats:bold> ) Edited barcode sequences are related to one another on the basis of shared mutations in order to reconstruct lineage trees. </jats:p> </jats:caption> <jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" orientation="portrait" position="float" xlink:href="353_aaf7907_fa.jpeg" /> </jats:fig> </jats:sec>

<jats:sec> <jats:title>BACKGROUND</jats:title> <jats:p>Materials by design is an appealing idea that is very hard to realize in practice. Combining the best of different ingredients in one ultimate material is a task for which we currently have no general solution. However, we do have some successful examples to draw upon: Composite materials and III-V heterostructures have revolutionized many aspects of our lives. Still, we need a general strategy to solve the problem of mixing and matching crystals with different properties, creating combinations with predetermined attributes and functionalities.</jats:p> </jats:sec> <jats:sec> <jats:title>ADVANCES</jats:title> <jats:p> Two-dimensional (2D) materials offer a platform that allows creation of heterostructures with a variety of properties. One-atom-thick crystals now comprise a large family of these materials, collectively covering a very broad range of properties. The first material to be included was graphene, a zero-overlap semimetal. The family of 2D crystals has grown to includes metals (e.g., NbSe <jats:sub>2</jats:sub> ), semiconductors (e.g., MoS <jats:sub>2</jats:sub> ), and insulators [e.g., hexagonal boron nitride (hBN)]. Many of these materials are stable at ambient conditions, and we have come up with strategies for handling those that are not. Surprisingly, the properties of such 2D materials are often very different from those of their 3D counterparts. Furthermore, even the study of familiar phenomena (like superconductivity or ferromagnetism) in the 2D case, where there is no long-range order, raises many thought-provoking questions. </jats:p> <jats:p>A plethora of opportunities appear when we start to combine several 2D crystals in one vertical stack. Held together by van der Waals forces (the same forces that hold layered materials together), such heterostructures allow a far greater number of combinations than any traditional growth method. As the family of 2D crystals is expanding day by day, so too is the complexity of the heterostructures that could be created with atomic precision.</jats:p> <jats:p>When stacking different crystals together, the synergetic effects become very important. In the first-order approximation, charge redistribution might occur between the neighboring (and even more distant) crystals in the stack. Neighboring crystals can also induce structural changes in each other. Furthermore, such changes can be controlled by adjusting the relative orientation between the individual elements.</jats:p> <jats:p>Such heterostructures have already led to the observation of numerous exciting physical phenomena. Thus, spectrum reconstruction in graphene interacting with hBN allowed several groups to study the Hofstadter butterfly effect and topological currents in such a system. The possibility of positioning crystals in very close (but controlled) proximity to one another allows for the study of tunneling and drag effects. The use of semiconducting monolayers leads to the creation of optically active heterostructures.</jats:p> <jats:p>The extended range of functionalities of such heterostructures yields a range of possible applications. Now the highest-mobility graphene transistors are achieved by encapsulating graphene with hBN. Photovoltaic and light-emitting devices have been demonstrated by combining optically active semiconducting layers and graphene as transparent electrodes.</jats:p> </jats:sec> <jats:sec> <jats:title>OUTLOOK</jats:title> <jats:p>Currently, most 2D heterostructures are composed by direct stacking of individual monolayer flakes of different materials. Although this method allows ultimate flexibility, it is slow and cumbersome. Thus, techniques involving transfer of large-area crystals grown by chemical vapor deposition (CVD), direct growth of heterostructures by CVD or physical epitaxy, or one-step growth in solution are being developed. Currently, we are at the same level as we were with graphene 10 years ago: plenty of interesting science and unclear prospects for mass production. Given the fast progress of graphene technology over the past few years, we can expect similar advances in the production of the heterostructures, making the science and applications more achievable.</jats:p> <jats:fig fig-type="figure" orientation="portrait" position="float"> <jats:caption> <jats:title>Production of van der Waals heterostructures.</jats:title> <jats:p> Owing to a large number of 2D crystals available today, many functional van der Waals heterostructures can be created. What started with mechanically assembled stacks ( <jats:bold>top</jats:bold> ) has now evolved to large-scale growth by CVD or physical epitaxy ( <jats:bold>bottom</jats:bold> ). </jats:p> </jats:caption> <jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" orientation="portrait" position="float" xlink:href="353_aac9439_fa.jpeg" /> </jats:fig> </jats:sec>