Available online at www.sciencedirect.com

ScienceDirect
DNA event recorders send past information of cells to
the time of observation
Soh Ishiguro1,2,3, Hideto Mori1,2,3 and Nozomu Yachie1,2,3,4,5
While current omics and single cell technologies have enabledmeasurements of high-resolution molecular snapshots of cells
at a large scale, these technologies all require destruction ofsamples and prevent us from analyzing dynamic changes inmolecular profiles, phenotypes, and behaviors of individual cells
in a complex system. One possible direction to overcome thisissue is the development of a cell-embedded ‘event recorder’system, whereby molecular and phenotypic information of a cell(s) can be obtained at the time of observation with their past
event information stored in ‘heritable polymers’ of the same cell.
This concept has been demonstrated by many synthetic cellular
circuits that monitor and transmit a certain set of environmental
and intracellular signals into DNA, and have now been further
accelerated by recent CRISPR-related technologies. Notably,
the discovery of the RT-Cas1–Cas2 system, which acquires
sequences of cellular transcripts into a specific host genomic
region, has enabled recording of a broader range of molecularprofile histories in the DNA tapes of cells, to understand the
dynamics of complex biological processes that cannot be
addressed by current technologies.
Addresses
1
Research Center for Advanced Science and Technology, The
University of Tokyo, Tokyo 153-8904, Japan
2
Institute for Advanced Biosciences, Keio University, Tsuruoka
997-0035, Japan
3
Graduate School of Media and Governance, Keio University, Fujisawa
252-0882, Japan
4
Department of Biological Sciences, School of Science, The Universityof Tokyo, Tokyo 113-0033, Japan
5
PRESTO, Japan Science and Technology Agency (JST), Tokyo
153-8904, Japan
Corresponding author:
Yachie, Nozomu (yachie@synbiol.rcast.u-tokyo.ac.jp)

Current Opinion in Chemical Biology 2019, 52:54–62
This review comes from a themed issue on Synthetic biology
Edited by Hirohide Saito and Yohei Yokobayashi

https://doi.org/10.1016/j.cbpa.2019.05.009
1367-5931/ã 2019 Elsevier Ltd. All rights reserved.

chromatin structures and transcriptomic, proteomic andmetabolomic profiles, as well as post-transcriptional andpost-translational modifications at a large scale. While thesetechnologies have uncovered how different biologicalsystems dynamically change their ‘average’ molecular
landscapes, recent single cell technologies have identified
many unknown but distinct types of cells in multicellularorganisms. Cells with different molecular phenotypes
dynamically emerge and orchestrate in the progression
and homeostasis of tissues, organs, and individuals. Therefore, it is extremely important to monitor how different
single cells (or clones) in such systems change their molecular profiles and interact with others. However, high content
molecular analyses of cells using current omics technologies
require destruction of samples and preclude performing
time course analysis of the same biological objects. For
example, it is extremely difficult to analyze how a given
blood cell clone of a mouse changes its molecular profiles
throughout the hematopoietic differentiation process.
Live cell imaging is capable of analyzing spatiotemporal
dynamics of molecules and cells with fluorescent proteinsor probes, but only for a limited number of objects, which canbe observed by microscopy (Figure 1a). Although single cell
technologies coupled with molecular barcoding of cells may
be a potential solution to obtain transcriptomic and (epi-)
genomic information of the same cell clones (or ‘close relative’
cells) across time course samples, the scalabilities of current
single cell technologies are up to the order of 105 cells per
sample, limiting the detection sensitivity to capture the same
barcoded cells across different samples (Figure 1b). Becauseof its high information capacity, physical density, durability,and ease of duplication, DNA has been proposed to store
artificial information [1], and several high capacity DNA data
storage methods have been accomplished [2–5]. In synthetic
biology, there is the concept of recording environmental andintracellular events in DNA that replicates with cell divisionsuch that the previous event histories of cells can be obtained
from the DNA media with other high content omics information at the time of observation (Figure 1c). Although noneof the current synthetic circuits have yet succeeded to record
high content, time course event information in cellular DNA
to address practical biological issues, the methodologies harnessing DNA recombination and CRISPR genome editing
have been rapidly heading toward this goal.

Introduction

Synthetic event recording using DNA
recombination

Massively parallel sequencing and mass spectrometry technologies obtain various types of cellular information, such as

To transmit extracellular event signals to permanentalterations of a DNA sequence, site-specific DNA

Current Opinion in Chemical Biology 2019, 52:54–62

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DNA event recorders Ishiguro, Mori and Yachie 55

Figure 1(a)Fluorescent probes(b)Time-course analysis of same barcoded clones
Single cell analysis

Barcoded clones

Barcoded clones

Barcoded clones

Barcoded cell population

Readout of past cellular statues from DNA tape(c)

DNA tape
Event
recordingEventrecording#321049

Cell #321049

Event
recording#321049

Cell #321049#321049

Cell #321049

Current Opinion in Chemical Biology

Different types of methods to analyze dynamic changes in molecular profiles of the same cells or clones. (a) Live cell imaging enables direct
observation of progressing cell populations by labeling target molecules with fluorescent markers, but only for a limited number of objects that canbe observed under a microscope. (b) Single cell transcriptome sequencing (scRNA-seq or single cell RNA sequencing) of barcoded cells enables
analysis of dynamic changes in transcriptome profiles. Cells in an initial population are uniquely tagged with DNA barcodes that can be identified
by an scRNA-seq pipeline. Clonal dynamics of transcriptomes can be observed for clones which barcodes can be observed across different timecourse scRNA-seq data. (c) The DNA event recorder concept. Each cell in a complex sample (e.g. a whole mouse body) involves a DNA event
recorder system that sequentially records cellular and environmental event information in a DNA tape. The past event history information of cells
can be derived from their DNA tapes with their high content omics profiles at the time of observation.

recombinases, such as Cre, Flp, PhiC31, and Bxb1, havebeen widely used with signal-inducible gene expression
promoters. In the simplest case, upon exposure to a
specific signal, a site-specific DNA recombinase isinduced in cells to delete or flip a given DNA sequence
sandwiched by two recombination sites, which can bethen readout, for example, by sequencing or as a restoration of fluorescent gene expression. Extensive efforts
have been made to screen orthogonal sets of recombinase-target site pairs, and to build more complex signaldependent circuits by concatenating multiple different
recombination sites and various promoters and terminators [6–10,11,12]. Examples include circuits that cancount the number of extracellular stimuli [6], circuits thatexhibit specific responses depending on the order ofdifferent stimuli [7], and various logic gate repertoires,such as AND and OR gates as well as NAND, NOR,
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XNOR, and others [8,9]. Another study has also reported
establishment of stringent band-path filters for precisioncomputation using multiple signal inputs [10]. Whilemost of these circuits have been demonstrated in Escherichia coli cells, BLADE (Boolean logic and arithmetic
through DNA excision) [11] enables flexible construction of various circuits in mammalian cells, which has
succeeded to construct 109 two-input, two-output circuits
in human embryonic kidney and Jurkat T cells; threeinput, two-output circuits; a six-input, one-output circuit;and circuits that incorporate CRISPR–Cas9 to regulate
endogenous genes.
In contrast to the fast-reacting DNA recombinase-based
methods with limited scalability owing to the number ofavailable recombinase enzymes as ‘DNA writer’ modules,
another scalable and slow-reacting method, SCRIBE
Current Opinion in Chemical Biology 2019, 52:54–62

56 Synthetic biology(synthetic cellular recorders integrating biological event),
has been developed using bacterial retron and recombineering technologies, which might be suitable to record
long-term histories of cells [13]. In this system, anengineered retron system is employed, whereby a chemical stimulus causes an arbitrary sequence harboredby common moiety sequences that form a specific
RNA secondary structure to be transcribed and reverse
transcribed by an RT enzyme. Thus produced singlestranded DNA (ssDNA) is then recombined with a target
DNA by homologous recombination enhanced by Beta
recombinase (from bacteriophage l). SCRIBE has been
demonstrated to successfully trace long-term exposure
patterns of multiple compounds in E. coli. The scalabilityof SCRIBE is high because orthogonal retron-target DNA
pairs can be freely designed by changing their donor
ssDNA sequences and target sites. Although the retron
system is originated from a bacterial species, it is known
to be also active in yeast [14] and mammalian cells [15].
Moreover, by harnessing CRISPR–Cas9 and the yeast
retron technology, CRISPEY (Cas9 retron precise parallel
editing via homology) has been established as a highly
efficient single base pair genome editing method in yeast[16], showing the potential of SCRIBE-type event recording in other eukaryotic cells.

CRISPR–Cas9 as a DNA writer module
Recently, CRISPR-related systems have started tobe employed for the DNA event recording concept. The
CRISPR–Cas system is a prokaryotic adaptive immune
system in which DNA sequences of foreign plasmids andphages are immunized into a genomic CRISPR (clusteredregularly interspaced short palindromic repeats) locus.
Upon second exposure to the same invaders, crRNAs(CRISPR RNAs) transcribed from the immunized
CRISPR locus recruit a Cas (CRISPR-associated) protein(s) to the target DNA molecules via sequence complementarity and digest them by the DNA cleavage activity of
Cas protein(s). Among the different types of CRISPR–Cas
immunity response systems, Cas9 from the type II-A
CRISPR system has been widely used for genome editing[17,18]. While a trans-activating crRNA (tracrRNA) is also
required to form a secondary structure with crRNA to
activate Cas9 at the target locus, a synthetic crRNAtracrRNA fusion, hereafter referred to as guide RNA(gRNA) [19], is commonly used in practical genome editing. In summary, CRISPR–Cas9 genome editing is a simple
two component system in which Cas9 with a doublestranded DNA cleavage activity is recruited by gRNA toa target DNA region containing a protospacer adjacentmotif (PAM) in its 30 -end. PAM is necessary for target
recognition by Cas9, conferring the distinction of theforeign target DNA and host crRNA-encoding region bothhaving the same sequence. Because DNA double-strand
breaks induce various DNA repair pathways, such as nonhomologous end joining (NHEJ) and homologous recombination (HR), CRISPR–Cas9 has been used to enhance
Current Opinion in Chemical Biology 2019, 52:54–62

gene knockout and knock-in efficiencies in model and nonmodel organisms.
Nuclease-deficient Cas9 (dCas9) and nickase Cas9
(nCas9) mutants can be tethered to various effector
protein modules (e.g. transcription activation complexes
and epigenetic modifiers) to enable synthetic control oftarget gene expression, epigenetic modifications, andchromatin structures [20,21]. Unlike previous synthetic
biology methods, this strategy allows flexible constructionof various synthetic circuits with minimal genetic engineering effort in various organisms. Furthermore,base editing technologies have been developed by fusing
Cas9 mutants to nucleoside-modifying enzymes, whichenables direct substitution of target nucleotides [22–24].
Among such technologies, fusing activation-induced cytidine deaminase (AID) and uracil glycosylase inhibitor(UGI) to nCas9 (or dCas9), highly efficient cytidine baseeditors for targeted C:G-to-T:A editing has been developed and improved by many groups [22,23,25–27](Figure 2a). In such targeted single base editing, gRNA
recruits AID and UGI to a target locus via nCas9 (ordCas9). AID converts cytidines in the target region touridines by deamination; and UGI blocks the glycosylation-initiated base excision repair pathway and enhances
the conversion of the uracil bases to thymine bases
through DNA replication [28]. While various applicationsof base editors have been proposed, such as in vivo
correction and creation of disease mutations [29–31],
several studies have also used them to construct DNA
event recorder circuits in cells.

Molecular event recording using CRISPR–
Cas9
Among the DNA event recording circuits based on
CRISPR–Cas9, mSCRIBE (mammalian synthetic cellular recorder integrating biological events) employed theconcept of self-targeting gRNA (stgRNA; see anotherarticle in this issue [32]) [33] and successfully recorded
combinatorial chemical conditions of human cell culture,in which two stgRNAs were regulated under the control
of doxycycline-inducible and IPTG-inducible promoters. The mSCRIBE concept was also used to establish
an inflammation-recording cell line using an NF-kBinducible Cas9 expression cassette and record lipopolysaccharide-induced inflammation levels of individual
mice in the synthetic DNA of the cells.
CAMERA (CRISPR-mediated analog multi event
recording apparatus) has been also proposed to constructsynthetic circuits that can transmit order information ofdifferent environmental signals into DNA [34]. In
CRIPSR–Cas9, the gRNA/target DNA heteroduplex
tends not to require perfect base pairing in the PAM
distal region, whereas the PAM-proximal ‘seed’ sequenceis necessary for the efficacy of Cas9 [35]. In CAMERA, by
harnessing this feature and using a C:G-to-T:A basewww.sciencedirect.com

DNA event recorders Ishiguro, Mori and Yachie 57

Figure 2

(a)

(b)
H2O

gRNA-1

NH3

S0

U

C

C
gRNA-2

UGI

S1
3’

5’
3’

M
PA

AID C

nCas9

PAM

Seed

T base editing
PAM

5’ GGGACCGGGTGGGAGTTTTGGTGGGACTGGATGGGG 3’
C

3’
5’

S2
5’

Seed

5’ GGGACCGGGTGGGAGCCCTGGTGGGACTGGATGGGG 3’

T base editing

5’ GGGATTGGGTGGGAGTTTTGGTGGGACTGGATGGGG 3’

gRNA
Output(c)Transcriptional
activation

BE-VP64*

Tandem repeat

GFP

**

GFP

*****

GFP

Current Opinion in Chemical Biology

Synthetic DNA circuits using CRISPR base editors. (a) Conceptual diagram of the cytidine base editor. (b) An example circuit demonstrated in the
CAMERA study. The S1-to-S2 state transition by base editing using gRNA-2 can be induced only after the S0-to-S1 transition by gRNA-1
generates the seed sequence for gRNA-2. (c) An example circuit demonstrated in the DOMINO study. The iterative domino-like editing of arepetitive sequence by a C:G-to-T:A base editor tethered to a transcription factor increases the fluorescent protein expression level over time.

editor, a circuit was established in E. coli cells, in whichdata recording by a secondary gRNA could not be
achieved until the first gRNA made a specific C:G-toT:A edit to create the seed sequence for the secondary
gRNA (Figure 2b). CAMERA was also used to record theintracellular status of mammalian cells. For example,
using the TCF/LEF promoter linked to the base editor,
lithium chloride-dependent recording of Wnt signaling
was achieved.
DOMINO (DNA-based ordered memory and iteration
network operator) employs the same concept used in
CAMERA, but achieves more complex circuits, in which,for example, an IPTG-inducible gRNA sequentiallyrestores the seed sequences of its three tandem overlapping
target sites so that the fourth editing by an arabinoseinducible gRNA can be made in accordance with theconcentration of IPTG and duration of IPTG exposure[36]. In this circuit, the sequential rounds of base editing
increase the copy number of targetable sequences for thesame gRNA. By fusing the transcription factor VP64 to thebase editor, a similar DOMINO circuit was also used todemonstrate an online state reporting circuit in mammaliancells, where the GFP expression level was increased alongwith time progression (Figure 2c).

CRISPR–Cas1–Cas2 as another class of DNA
event recorder systems
While CRISPR immunity systems are evolutionary
diverged, one of which is Cas9 from the type II-A
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CRISPR system, the upstream CRISPR immunization
process is relatively conserved across species, in which theCas1–Cas2 protein complex is commonly involved in theacquisition of invasive DNA into the CRISPR locus(Figure 3a) [37–39]. The genomic CRISPR sequence
consists of a leader sequence followed by an array of
periodic repeats interspaced by previously immunizedspacer sequences (as its acronym stands for) and theimmunization of a new ‘protospacer’ DNA always occurs
on the leader-proximal repeat, creating a new interspaced
repeat unit (Figure 3b). Therefore, the order of spacers in
the CRISPR array from the tail to head direction can beseen as an old-to-new historical immunization or invasion
record of foreign DNA fragments in the host cell.
Active recording of high content artificial information into
an E. coli cell population was recently demonstrated using
the type I-E CRISPR acquisition system [40]. In thisstudy, three sets of artificial protospacers were preparedseparately for five rounds of information recording. For
each round every 20 hours, three protospacers each from adifferent set were pooled and introduced into the cell
population. Although a single bacterial cell cannot efficiently record all protospacer queries in its CRISPR locus,
newly queried protospacers are always acquired in the
upstream region of the older spacers. Using this property,
a computational framework enabled prefect reconstruction of the orders of protospacer queries for all three setsfrom the massively parallel sequencing data of the
population’s CRISPR arrays. This approach was also
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58 Synthetic biology

Figure 3

3’

5’

(b)

PA
M

(a)

PAM

Cas1–Cas2 complex

Direct repeats
5’3’

Cas2 dimer

Leader3’
5’Previously acquired spacers

PA
M

Cas1 dimers(c)

Input signal
1st nucleophilic attack

Constant spacer acquisition
PAM

Cas1–Cas2 complex

Leader

2nd nucleophilic attack

Plasmid

Trigger DNA
Signal-dependent
spacer acquisition

PA
M

GenomeUnidirectional recording

180°

DNA repair

CRISPR array
Newly acquired spacer
Current Opinion in Chemical Biology

Spacer acquisition by CRISPR–Cas1–Cas2. (a) Structure of the Cas1–Cas2 protein complex bound to a protospacer DNA (PDB number: 5DQZ).
(b) Schematic representation of spacer acquisition to a CRISPR array by the Cas1–Cas2 complex. (c) Conceptual diagram of TRACE. The copy
number of the trigger plasmid is induced by a signal input, by which the spacer acquisition from the trigger plasmid is also enhanced, while thatfrom the reference DNA remains at the same level.

demonstrated in another study, in which the world’s first
movie ‘The Horse in Motion’ by Edward Muybridge in
1878 was converted into a five-frame 36  26 pixel animation and recorded in a cell population using a pool of
104 protospacers for each frame [41]. The five sets of thecomplex protospacer pools were sequentially introduced
into a cell population from which more than 90% of themovie data were recovered.

Temporal recording of extracellular signals
using Cas1–Cas2
The CRISPR–Cas1–Cas2 system can be also used torecord environmental event histories of cells in their
CRISPR arrays. As seen in high content artificial data
recording examples, the directionality of spacer acquisition in the CRISPR locus allows simple implementationof a synthetic circuit to record time course event signals.
TRACE (temporal recording in arrays by CRISPR expansion) is the first example that enables recording of temporal environmental stimuli of an E. coli cell population
into their CRISPR arrays using CRISPR–Cas1–Cas2[42]. This method employs the concept of a trigger
plasmid (pTrig), in which the phage P1 lytic replication
Current Opinion in Chemical Biology 2019, 52:54–62

protein RepL and its replication origin are placed downstream of a chemically inducible promoter, so that the
copy number of the plasmid in cells can be elevated by
the corresponding environmental compound. As opposedto the pTrig plasmid, the other cellular DNA, such as
genomic DNA and the other plasmid encoding the anhydrotetracycline (aTc)-inducible Cas1–Cas2 system, is relatively stable and serves as reference DNA. While theacquisition of spacers from pTrig and the reference DNA
occur at a constant rate when aTc induces Cas1–Cas2, thespacer acquisition frequency from pTrig is elevated whencells are also exposed to its corresponding chemical signal(Figure 3c). In TRACE, four variants of pTrig plasmids
have been developed for IPTG, cooper, trehalose, andfructose signals, allowing time course event recording of
combinatorial signal patterns into CRISPR arrays of a cell
population at a certain degree.

Transcriptome-scale molecular recording intocell populations using RT-Cas1–Cas2
A common drawback in all of the abovementioned systems
is that the recording targets need to be predefined in thecircuit and are restricted to a limited number and kinds of
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DNA event recorders Ishiguro, Mori and Yachie 59

chemical compounds or cellular events that can be linked to
specific promoters available in a target system. The ideal
DNA event recorder would autonomously and efficiently
record a wider range of temporal molecular and environmental information of cells to trace a variety of cell statuses.
Because type III CRISPR systems have activities todigest RNA [43–46] and some of their Cas1 genes arenaturally fused to reverse transcriptase domains (RTCas1) in various species [47–49], it has been suggested
that there is a conserved mechanism of spacer acquisition
from RNA such as for the defense against RNA viruses.
The first example of RNA-derived spacer acquisition has
been recently discovered in Marinomonas mediterranea(MMB-1), in which intracellular RNAs are reverse
transcribed and acquired in the genomic CRISPR arrayby the RT-Cas1–Cas2 complex [50]. While functional
reconstitution of MMB-1 RT-Cas1–Cas2 to yet to be
performed in E. coli, another recent study has demonstrated that Fusicatenibacter saccharivorans RT-Cas1–Cas2(FsRT-Cas1–Cas2) can work heterologously in E. coli toacquire sequences of both endogenously expressed RNAsand viral RNAs into a synthetic CRISPR array. By developing a method to efficiently capture newly expanded
CRISPR spacers, namely SENECA (selective amplification of expanded CRISPR arrays), for massively parallel
sequencing, this study also established the Record-seq
method that enables transcriptome-scale molecular
recording in cell populations (Figure 4) [51].
In this study, Record-seq successfully captured RNAderived spacer acquisition from a chemically inducible
transgene by which the acquisition frequency in the

populational CRISPR array sequences was also elevatedalong with the increased input level of the chemical compound. Furthermore, while transcriptome and Record-seq
profiles of E. coli cells in each of the different growth phases
correlated at a certain level, a higher correlation wasobserved between a transcriptome profile of the log phaseand a Record-seq profile of the stationary phase, showingthat Record-seq captured past molecular profile informationof cells observed at the time of the experiment. Record-seq
also distinguished whether cell populations were underoxidative (or acid) stress or not, and whether cell populations
were transiently experiencing herbicide exposure, which
could not be differentiated by their transcriptome profiles.
This is the first demonstration of a synthetic DNA recordersystem that enabled retrospective reconstruction of past cell
population statuses by recording global molecular profiles
without defining specific target events.
Because SENECA identifies newly acquired spacer units
separately, it currently limits the capacity of the Recordseq method that can readout only an additive (or cumulative) transcriptome profile for some ambiguous durationin the past of the cell population without their time course
information. However, similar to the other Cas1–Cas2based recording methods that successfully reconstruct theorder of different event signals from CRISPR arrays,
further developments would enable serial recording ofdynamic changes in transcriptome profiles into cellular
CRISPR arrays.

Outlook and discussion
The permanent recoding of environmental stimuli into
cellular DNA has been achieved by synthetic circuits

Figure 4

E. coli genome

Transcriptional recording plasmid
PT7lac
RT-Cas1

LeaderCas2
Direct repeat
Heterologous
expression

RT-Cas1–Cas2
protein complex
Intracellular transcriptome
RT-Cas1–Cas2–RNA

RNA acquisition(RNA to DNA conversion)
Newly acquired spacer

Parental CRISPR array

Transcriptional record stored in DNA

Current Opinion in Chemical Biology

Recording of a bacterial transcriptome in a synthetic CRISPR array by RT-Cas1–Cas2.
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Current Opinion in Chemical Biology 2019, 52:54–62

60 Synthetic biology

using DNA recombinases and more recently by CRISPRrelated approaches. However, all of the current methods
remain at the proof-of-concept stage, which have demonstrated molecular recording of event histories only at thecell population level. None of them have yet accomplished analysis of heterogeneous cells or clones withdiverse phenotypic trajectories such as differentiation.
This is because the numbers of target signals that canbe efficiently recoded in synthetic DNA tapes have been
currently limited to distinguish diverse cell statuses, and/or the signal-to-memory transmission efficiencies of the
DNA writer modules have been too low to record sufficient amount of information in DNA. While mSCRIBE,CAMERA, and DOMINO have been demonstrated toachieve highly efficient recording of cellular events insynthetic DNA introduced into mammalian cells, thesignal events that can be recoded simultaneously in thesesystems have been limited to those with available corresponding promoters. Although RT-Cas1–Cas2 has thepotential to record transcriptome profiles into DNA, its
spacer acquisition efficiency is extremely low [51].
Furthermore, neither Cas1–Cas2 nor RT-Cas1–Cas2
has been successfully reconstituted heterologously ineukaryotic cells.
The DNA event recorder concept would be useful tostudy heterogeneous progression of mammalian systems
only when it renders recording of a broader range of past
information in DNA tapes at the single cell resolution orat least at the level of cell type groups, each of whichcorresponds to a distinct cell progression trajectory. Whilethe establishment of RT-Cas1–Cas2-based molecular
recording in mammalian cells could be one of the goals,
it would require discovery of a new RT-Cas1–Cas2 variant that can heterologously work in mammalian cells or
cryptic bacterial factors that support the function of thepreviously characterized RT-Cas1–Cas2 in mammaliansystems, as well as enhancement of their efficiencies.
Therefore, another possible direction could be increasing
the scalability of Cas9-based approaches. In the inflammation recording by mSCRIBE and the Wnt signaling
recording by CAMERA, the gene expression of the DNA
writer modules, Cas9 or the C:G-to-T:A base editors, wasunder the control of specific promoters. However, a better
approach would be regulating expression of multiple
gRNAs in accordance with their respective promoterslinked to specific gene expression. While gRNAs cannot
be naturally expressed from variety of RNA polymerase
type II (pol II) promoters, recent studies have shown thatgRNAs concatenated with self-cleaving ribozymes [52] ortRNAs [52,53] can be expressed by pol II promoters.
Furthermore, riboswitch systems have been demonstrated to regulate folding of the mature gRNA secondary
structure in response to a ‘trigger’ RNA [54], showing thepotential to construct large RNA circuits that transmit abroader range of RNA expression patterns to synthetic
DNA tapes. For example, embryonic stem cells harboring
Current Opinion in Chemical Biology 2019, 52:54–62

a circuit that enables simultaneous recording of Oct4,
Sox2, Klf4, cMyc, and other maker gene expression
histories would be of great use in studying cell differentiation and reprogramming processes.
Taken together, the current progress in this field suggests
some potential approaches to establish high capacity cellembedded DNA event recorder systems that overcome
the limitations of the current technologies to tracechanges in molecular profiles and environmental conditions of different cells in complex biological systems
including mammalian tissues and individuals. Moreover,as discussed in another article of this issue [32], thedevelopment of ‘evolving DNA barcodes’ has also been
rapidly accelerated for high resolution cell lineage tracingof multicellular organisms with the concept of DNA
barcodes and CRISPR–Cas9 to establish a new framework to discuss heterogeneous cellular phenotypes in thecontext of cell lineages. Further developments in both ofthese fields would enable us to understand how mammalian and other heterogeneous systems develop and function with their molecular and cellular contexts.

Conflict of interest statement
Nothing declared.

Acknowledgements
We thank members of the Yachie laboratory for discussions about different
aspects of the current and past cell lineage tracing approaches. The research
projects of the Yachie laboratory in this field are supported by grants fromthe Japan Science and Technology Agency (JST) PRESTO and CREST
programs, the New Energy and Industrial Technology Development
Organization (NEDO) Smart Cell Project, the Japan Agency for Medical
Research and Development (AMED) PRIME program, the Shimadzu
Science Foundation, the Takeda Science Foundation, the SECOM Scienceand Technology Foundation, the Naito Foundation, the Nakajima
Foundation, and the Asahi Glass Foundation.

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