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.

Figure 1.. Components of cellular memory.

a | Cellular recording devices can be engineered into multicellular organisms or unicellular populations, and their general architecture can be broken down into four major components: signal sensing, DNA writing, DNA reading and actuation. b | Properties or examples of each of the four major components. AbR, antibiotic resistance; ssDNA, single-stranded DNA.

Figure 2.. Example DNA recording devices.

The functionality of four exemplary DNA-based recorders are illustrated following the recording device architecture. a | Recombinase state machine (RSM) fixed-address writer. Orthogonal recombinases are expressed in response to a signal, and they mediate excision or inversion events at a designed recombinase address (filled triangles are unrecombined sites, unfilled triangles are recombined sites). Based on the ordering of inputs, different resulting address sequences can be achieved, which are read out by sequencing, or which can mediate functional responses by interleaving genetic parts (promoters, expression cassettes, or terminators) within the recombinase address. b | CRISPR-mediated analog multi-event recording apparatus (CAMERA) flexible writer. Single-guide RNAs (sgRNAs) are expressed in response to a signal, and direct a base editor (dCas9-BE) to mutagenize specific loci within a genomic address, which can be read out by sequencing. c | Mammalian synthetic cellular recorder integrating biological events (mSCRIBE [G]) stochastic writer. A self-targeting guide RNA (stgRNA) is expressed in response to an input signal, and directs Cas9-mediated editing and generation of a small insertion or deletion (indel) at the same stgRNA address, resulting in continuous editing and sequence evolution. The resulting stgRNA address can be read out by sequencing. d | Temporal recording in arrays by CRISPR expansion (TRACE) directional writer. A signal is converted into altered DNA abundance through use of a copy-number inducible trigger plasmid (pTrig). Short spacers can be incorporated into a genomic array address in a directional manner, either from the trigger sequence or at a constant rate from genomic or plasmid reference sequences. Resulting arrays can be sequenced and the order and source of spacers can be compared to a model of CRISPR expansion to classify the signal input sequence over time.

Figure 3.. Applications of DNA-based biological recorders.

a | Use cases of DNA based recording (top) as well as applications across research and applied settings (bottom). b | Example utility of DNA-based recorders in the gut microbiome. Engineered cellular memory devices could be utilized for noninvasive multiplex temporal recording of important signals such as nutrient status and microbial- and host-derived metabolites. In addition, these recorders could mediate functional actions in response to specific signals or profiles of inputs. SCFA, short-chain fatty acids.