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DNA-based memory devices for recording cellular events

Abstract

Measuring biological data across time and space is critical for understanding complex biological processes and for various biosurveillance applications. However, such data are often inaccessible or difficult to directly obtain. Less invasive, more robust and higher-throughput biological recording tools are needed to profile cells and their environments. DNA-based cellular recording is an emerging and powerful framework for tracking intracellular and extracellular biological events over time across living cells and populations. Here, we review and assess DNA recorders that utilize CRISPR nucleases, integrases and base-editing strategies, as well as recombinase and polymerase-based methods. Quantitative characterization, modelling and evaluation of these DNA-recording modalities can guide their design and implementation for specific application areas.

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Acknowledgements

The authors apologize to colleagues whose work could not be cited owing to space limitations. H.H.W. acknowledges funding from the US National Institutes of Health (1R01AI132403-01), the US Office of Naval Research (N00014-17-1-2353, N00014-15-1-2704), the US National Science Foundation (NSF; MCB-1453219) and the Burroughs Wellcome Fund Pathogenesis of Infectious Disease (PATH; 1016691). R.U.S. is supported by a Fannie and John Hertz Foundation Fellowship and an NSF Graduate Research Fellowship (DGE-11-44155).

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Nature Reviews Genetics thanks T. Fulga, Y. Liu and Y. Michaels for their contribution to the peer review of this work.

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Both authors contributed to all aspects of the manuscript.

Correspondence to Harris H. Wang.

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Glossary

Cas1–Cas2 CRISPR integrase

Conserved machinery in CRISPR immune systems mediating integration of short spacers from intracellular DNA sources into genomic arrays in a directional manner.

Site-specific recombinase systems

Systems composed of a recombinase enzyme and flanking target recognition sites around a target sequence. These systems enable inversion, excision or integration of the target sequence on the basis of the orientation of recognition sites.

Recombinase state machine

(RSM). A fixed-address writer encompassing a formalized architecture of genetic programmes created from combinations of three orthogonal recombinase systems.

Synthetic cellular recorder integrating biological events

(SCRIBE). A single-stranded DNA (ssDNA)-recombination-based flexible writing approach.

Retron

A bacterial reverse transcriptase system that produces a molecule that is a hybrid of RNA and single-stranded DNA (ssDNA) called multicopy ssDNA (msDNA).

mSCRIBE

(mammalian SCRIBE). A Cas9-nuclease-based stochastic writing approach.

CRISPR-mediated analog multi-event recording apparatus

(CAMERA). A base-editing-based flexible writing approach.

Base editing

A Cas9-based genome engineering approach in which a catalytically dead Cas9 (dCas9) with no nuclease activity is linked to a deaminase (dCas9-BE), enabling single-base-pair genomic mutation at desired locations.

Catalytically dead Cas9

(dCas9). A modified version of Cas9 that lacks endonuclease activity via engineered point mutations. It can be linked to other effector domains for diverse sequence-specific genome engineering applications.

Cas9

CRISPR-associated protein 9; a genome engineering nuclease tool enabling cleavage of desired genomic sites specified by a single-guide RNA (sgRNA).

Non-homologous end joining

(NHEJ). An endogenous pathway enabling repair of double-strand breaks (DSBs).

Self-targeting gRNA

(stgRNA). A single-guide RNA (sgRNA) that is targeted to its own sequence, which enables stochastic sequence evolution over time.

Directional writers

DNA writing relying on directional addition of single or multiple base pairs.

DNA polymerase

A type of enzyme that replicates DNA polymers on the basis of an existing template DNA by serial addition of individual nucleotides.

Temporal recording in arrays by CRISPR expansion

(TRACE). A Cas1–Cas2-based CRISPR spacer acquisition system to record biological signals over time.

Fluorescence resonance energy transfer

(FRET). A biochemical mechanism of energy transfer between two chromophores that can be utilized for sequence-specific DNA detection applications.

Memory by engineered mutagenesis with optical in situ readout

(MEMOIR). A Cas9-nuclease-based stochastic writing approach with spatial readout by single-molecule RNA fluorescence in situ hybridization (smFISH).

Genome editing of synthetic target arrays for lineage tracing

(GESTALT). A Cas9-nuclease-based stochastic writing approach enabling large-scale lineage tracing applications.

Terminal deoxynucleotidyl transferases

(TdTs). DNA polymerases that can add nucleotides to DNA without a template.

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Further reading

Fig. 1: Components of cellular memory.
Fig. 2: Examples of DNA-recording devices.
Fig. 3: Applications of DNA-based biological recorders.