MOLECULAR BIOLOGY

Molecular recorders to track cellular events
DNA tapes could be used to record dynamic molecular and cellular events in animals
By Nanami Masuyama1,2, Naoki Konno3,Nozomu Yachie1,4S

1School of Biomedical Engineering, Faculty of Applied
Science and Faculty of Medicine, The University of British
Columbia, Vancouver, BC, Canada. 2Institute for Advanced
Biosciences, Keio University, Yamagata, Japan. 3Departmentof Biological Sciences, Graduate School of Science, The
University of Tokyo, Tokyo, Japan. 4Research Center for
Advanced Science and Technology, The University of Tokyo,Tokyo, Japan. Email: nozomu.yachie@ubc.ca
SCIENCE science.org

Altering the type of information-writing
enzyme would also increase the information
recording capacity. Most of the current celllineage tracing systems use wild-type Cas9,
which induces double-stranded DNA breaks
that are toxic to cells and can also confer large
deletions, especially when multiple breaks
are introduced simultaneously. Additionally,
these large deletions can overwrite acquired
mutations and progressively collapse the barcodes. These limitations could be alleviatedby using a CRISPR base editor (BE), fusing
a catalytically inactive Cas9 to a nucleosidedeaminase that introduces single-nucleotide
substitutions at the gRNA target sequencewith minimal double-stranded DNA breaks.
Most BEs confer unidirectional substitutions(e.g., C T or A G) (7). This could be a downside, given that the lineage recording ability
crumbles when all substrate C or A bases inthe DNA barcodes are converted and thus depleted. However, the recent development ofdual-function BEs has enabled the simultaneous introduction of C T and A G substitutions. Using such a dual-function BE thatcan confer reversible C:G T:A mutations
will enable more-accurate lineage tracing
than wild-type Cas9 or single-function BEs.
In evolutionary biology, phylogenetic trees
have been efficiently reconstructed from revertant mutations. BEs have also been demonstrated to record cellular signals but only
for a limited number of events in mammaliancells, such as Wnt signaling (8).
The CRISPR prime editor (PE), fusing acatalytically inactive Cas9 to reverse transcriptase, has recently been proposed for
scalable, high-content event recording. In
these genome-editing systems, a PE gRNA(pegRNA) encodes a synthetic short payload
sequence and recruits the PE to the target
sequence, in which—coupled with endogenous DNA repair machineries—the reverse
transcription activity of PE enables the integration of the payload sequence to the target
site (7). This enables more diverse data encoding per barcode than wild-type Cas9 andBEs. Preliminary data show that the gene
expression activities of 300 enhancers can berecorded to a single target DNA locus of a cell
population using a library of pegRNAs, eachof which is encoded downstream of a specificenhancer element (9). PE has also been programmed for a cascade reaction, where theorder of complex events can be recorded byarraying event-specific payload sequences
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tarting from a fertilized egg, cells proliferate, pass their genomic information to their progeny, and dynamicallychange their functions to form diverse
tissue structures. Throughout development, intracellular and environmental
cues trigger patterns of gene expression thatgovern cell state transitions and produce further cellular and environmental cues, leading
cells to self-organize into functional clusters
in spatially distinct areas. How can these processes be investigated? High-resolution molecular snapshots of cells can be obtained byvarious omics technologies, but these require
sample destruction, which precludes analyses
over time. Live-cell imaging with fluorescentprobes can analyze time-course dynamics but
is limited to a small number of molecules orcells. These challenges could be overcome by
DNA event recording, where molecular andcellular information is progressively stored
in synthetic “DNA tapes” in cells within amulticellular organism, and the stored information is accessed by high-throughput DNA
sequencing. However, there are technicallimitations that need to be overcome.
One of the DNA event recording modalities, dynamic cell lineage recording using
CRISPR-Cas9 genome editing, has accelerated developmental cell lineage tracing. In
this system, chromosome-embedded synthetic DNA barcode arrays are continuously
and randomly mutated by Cas9, which isrecruited by the expression of many barcodetargeting guide RNAs (gRNAs); these mutated arrays are progressively inherited bydaughter cells (1–3) (see the figure). Similar
to an evolutionary tree reconstruction fromgenomic sequences of diverse species, thecell lineage of an animal initiated from a
fertilized egg can be reconstructed according to the mutation patterns of the barcode
sequences in descendant cells at the time ofobservation using high-throughput DNA sequencing. Such lineage tracing systems havealso been linked to single-cell RNA sequencing (scRNA-seq), where individual cell types
identified by scRNA-seq can be linked totheir historical cell lineage information (3).
Today, new technologies are rapidly revising perspectives on developmental biology, such as the origins of the hypoblast andembryo-restricted epiblast (4), blood cell differentiation, and the deciphering of cell proliferation dynamics of metastatic cancers (5,6). Furthermore, ideas to develop other cellembedded circuits that can convert environmental and cellular signals to genome-editing
activities have emerged (2). Combined witha cell lineage recorder, such systems wouldenable the computational reconstruction andmapping of historical cellular events and cellstate changes onto a cell lineage. These ideascould lead to the characterization of highresolution cell lineages, cell state changes,
and cell communications during whole mammalian body development, homeostasis, andmalfunction. This would deepen knowledge
about developmental and cancer biology that
could be translated into therapeutic strategies. However, DNA event recording has yet
to trigger the next gold rush in biology because there are some missing components.
The current molecular event recording methods need to overcome four substantial limitations in information recording capacity, available molecular sensors to capture diverse
biological events, the number of cells that canbe analyzed, and computational capacity toreconstruct high-content biological history.
Information recording capacity could beincreased by extending the total length of theDNA barcodes. For example, several lineage
tracing methods have used multiple short
DNA barcode units per cell for the random
accumulation of CRISPR-induced mutations.
To read out the combinatorial mutation patterns in these barcodes in distal genomic loci,constitutively active promoters are introduced to allow transcription, and the mutation information is captured by scRNA-seq.
The current methods use either a few copies
of long gRNA-targeting barcode arrays (up to10 barcodes per array, whose size is restrictedby short-read DNA sequencing methods) ormany copies of short barcode arrays withsingle gRNA-targeting units. There seems
to be no barrier to combining these two approaches to achieve a high-content event
recording system (i.e., encoding many longbarcode arrays per cell).

INSIGHTS | P E R S P E C T I V E S

to capture the whole developmental processof an animal body.
High-performance computing methodsalso need to be developed with these engineering efforts. Although many of thecurrent cell lineages have been generated
by repurposing existing evolutionary tree
reconstruction tools, their algorithms need
to be optimized for information recording
biases in specific lineage tracing methods(13). Efficient algorithms for other biological events have yet to be established.
Furthermore, no current phylogeny estimation software tool can reconstruct a lineageof more than one million inputs, raisingconcerns about how to reconstruct a biological history forming a mammalian body

The DNA event recording concept

CRISPR DNA recorders

Alterations

Engineered cellSignals

3'
5'

5'
3'

Cas9Editing

DeletionsgRNA

Geneexpression*Deaminase
C5'
3'Baseeditor

3'
5'

Array
ID

Random editing**gRNA

3'
5'

Prime
editorBarcodearray

Substitutions(C T or A G)
Lineagetracer

Reversetranscriptase
5'
3'

*

Eventrecorder

*
**

**
*
*

Payloadinsertion

REF ER ENCES AND NOTES

pegRNAgRNA, guide RNA; pegRNA, prime editor gRNA.

the advent of the droplet-based methods thatenabled the analysis of multiple thousands ofcells (11), it is not unreasonable to predict thattechnologies that can capture a greater magnitude of cells will appear in the next 5 years.
Additionally, spatial RNA-seq has enabled
spatial profiling of gene expression patterns
in a tissue section or a three-dimensional
block in high resolution (12). Coupled withDNA event recording systems whose readout
information is converted into RNA, this modality also enables the reconstruction of celllineage and cell state trajectories throughouta physical space. However, spatial RNA-seq
allows only the reconstruction of later events
that contributed to its formation. Therefore,
more-scalable methods need to be developed
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29 JULY 2022 • VOL 377 ISSUE 6605

of at least hundreds of millions of cells. Anew computing framework called deep distributed computing has been proposed toovercome this limitation (14). This framework has enabled accurate cell lineage
tracing of more than two hundred million
mutated sequences. In brief, starting froma single computing node, it reconstructs an
upstream lineage and assigns all the input
sequences into its subclades, where an independent computing node recursively resolves the next upstream lineage of eachsubclade. Like a spreading virus, a single
computing task autonomously generates
many distributed computing tasks, each ofwhich iterates the same procedure. Thereis no conceptual barrier to implementing

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Cell-embedded synthetic CRISPR-Cas information-writing enzymes can progressively record cellular events andlineage transitions into DNA tapes (or barcode arrays) by introducing various types of DNA alteration. DNA tapes areread out by single-cell sequencing, and the biological event histories are computationally reconstructed. Cell lineage
can be recorded by continuous accumulation of random mutations into DNA tapes. Other event recording systems
necessitate editing event-specific DNA tape positions or inserting specific DNA payloads into the DNA tape.

other event reconstruction processes into
this framework.
Technologies for high-resolution DNA
event recording have been maturing, which
could expand the horizons of biology in different animal models—perhaps starting frommice for mammalian species. It is expectedthat this approach will identify unknown celldifferentiation behaviors and their underlying molecular and cellular regulatory mechanisms. Analysis of many individuals’ developmental processes in a single species in highresolution would characterize deterministic
and stochastic cell differentiation patterns
and quantify the ability of cells to undergo
state transitions, enabling more precise computational modeling of dynamic multicellular systems with biophysical perspectives.
Direct observation of human developmental
procedures with the same approach is impossible because of ethical considerations
regarding the genetic manipulation of human embryos. However, high-resolution developmental maps of diverse nonhuman species and comparative genomics approaches
could also reveal human developmental
processes. Furthermore, as seen in the emerging CRISPR cell lineage tracing studies
of human organoid development (15) andtumor progression to metastasis in a mousemodel (5, 6), embedding a high-content DNA
event recorder system in human pluripotent
stem cells for organoid studies and in cancer
cells for animal xenograft studies can more
directly contribute to understanding theirbehaviors. Combined with genome-editing
technologies that can reconstitute disease
mutations in the human genome, these newmodel systems will also serve as testbeds
with substantially expanded data monitoring
abilities in therapeutic development for genetic disorders and cancers. j

ACKNOWL EDGMENTS

We thank members of the Yachie laboratory for useful
insights, especially S. King and A. Adel. The authors aresupported by the Canada Research Chair program, a project
grant (PJT 178040) by the Canadian Institutes for HealthResearch, and a Pilot Innovation Fund (PIF003) by Genome
British Columbia. N.M. and N.K. are supported by fellowships
from the Japan Society for the Promotion of Science. N.M. andN.K. contributed equally to this work.
10.1126/science.abo3471

science.org SCIENCE

GRAPHIC: K. HOLOSKI/SCIENCE

at a target DNA locus like a ticker tape (10).
However, these approaches have not yet been
implemented in animal models.
High-content event reconstruction necessitates high-capacity scRNA-seq. A cell lineage
recording system that confers at least one
unique mutation per cell division would yield
the perfect reconstruction of cell lineage if allcells of a body can be analyzed. Analysis of alimited number of cells enables the historicalreconstruction of only an upstream cell lineage whose descendant information can becovered by the analytes. The current state-ofthe-art scRNA-seq can process multiple millions of cells, whereas a mouse body consistsof multiple billions of nucleated cells. Given
the rapid development of scRNA-seq sinceMolecular recorders to track cellular events
Nanami MasuyamaNaoki KonnoNozomu Yachie

Science, 377 (6605), • DOI: 10.1126/science.abo3471

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