Starting from a fertilized egg, cells proliferate, pass their genomic information to their offspring, and dynamically change their functions to form diverse tissue structures. Throughout development, intracellular and environmental cues trigger patterns of gene expression that govern cell state transitions and produce additional cellular and environmental cues, leading cells to self-organize into functional clusters within spatially distinct areas. How can these processes be investigated? High-resolution molecular snapshots of cells can be obtained using various omics technologies, but these methods require the destruction of the sample, thereby precluding time-course analyses. Live-cell imaging with fluorescent probes can analyze time-course dynamics but this method is limited to analyzing only a small number of molecules or cells. To overcome this common obstacle in biology, our research program is pioneering two major fileds in biology: DNA event recording and retrospective clone isolation.

Keywords:
Synthetic Biology, Computational Biology, Developmental Biology, Stem Cell Biology, Mouse Engineering, High-Performance Computing

In the idea of DNA event recording, molecular and cellular events of a multicellular organism are progressively stored in synthetic “DNA tapes,” like a molecular ticker tape (Science 2022). Such a system allows for the readout of historical molecular expression profiles of many cells using high-throughput single-cell sequencing. Analogous to a video camera system, the requirements of DNA event recording systems are: (1) high-capacity DNA “memory” modules embedded in chromosomes; (2) highly sensitive “sensor” modules to capture cell divisions and cell types; (3) information “writer” modules to alter DNA tapes; and (4) high-capacity information “reader” modules to reconstruct complex biological history information written in DNA tapes.

Another idea to achieve dynamic, high-content observation of progressive systems is retrospective clone isolation. In this concept, cells in a population are first tagged with unique, short DNA barcodes and propagated. A subpopulation is then subjected to a specific assay. After identifying a barcode for a clone of interest, the same clone, or its close relatives, is isolated in a barcode-dependent manner from the initial population or any other subpopulation stored during the experiment. The isolated clone can be subjected to various experiments, including omics measurements and synthetic population reconstitution assays. We recently established a multi-kingdom, highly efficient retrospective clone isolation method using CRISPR base editing (bioRxiv 2022). We are currently working on deciphering the molecular priming factors that determine cell fates in different human pluripotent stem cell differentiation and organoid models.

To achieve these visions, we have incrementally worked on diverse topics in biology.

In CRISPR-Cas9 genome editing, a guide RNA recruits Cas9 to a target genomic region adjacent to a protospacer adjacent motif (PAM) sequence, 5’-NGG-3’, and Cas9 produces a DNA double-stranded break (DSB). This event promotes either gene deletion or transgene insertion through the induction of different DNA repair pathways, but DSBs are cytotoxic and the DSB-based editing outcomes are unpredictable. In collaboration with Dr. Keiji Nishida, we developed a new genome-editing tool, Target-AID, which fused cytidine deaminase (AID) to a nickase Cas9 and enabled highly precise targeted C→T substitutions without DSBs (Science 2016). In collaboration with Dr. Osamu Nureki, we also succeeded in increasing the targeting scope of Cas9 from a restrictive NGG to an NG PAM (Cas9-NG) and developed Target-AID-NG (Science 2018). Furthermore, my team developed a new base editor Target-ACEmax, which enables the simultaneous induction of C→T and A→G substitutions on a target DNA molecule and greatly expands the potential of base editing in therapeutics and biotechnology development (Nature Biotechnology 2020). Furthermore, to explore new genome editing tools apart from the CRIPSR-Cas9 and other characterized genome editing tools, we have developed a tool to rapidly capture periodic and interspaced periodic repeats from genomic and metagenomic resources (Nucleic Acids Research 2019).

Several methods have been proposed to trace developmental cell lineages of multicellular organisms, whereby chromosome-embedded DNA barcodes are continuously mutated by Cas9 and inherited from mother to daughter cells, whose lineage can be reconstructed according to the mutation patterns at the time of observation. However, none of these technologies has achieved high-resolution lineage tracing. To decipher the map of the whole-body mammalian (mouse) developmental process at the single-cell resolution, we have outlined the key issues and perspectives in this field (Science 2022) and are developing new genetic circuits, mouse engineering, and high-performance computing technologies. Target-AID and Target-ACEmax, described above, were developed primarily for the purpose of high-resolution cell lineage tracing. We also developed a new deep distributed computing platform and succeeded in the precise lineage reconstruction of over 235 million mutated sequences generated by a simulator (Nature Biotechnology 2022).

Questions such as "Did the chemotherapy-resistant clones exist in the initial cell population with a unique cellular status from the beginning?" or "Did any molecular factor underlie the observed stem cell differentiation fates?" highlight many unresolved biological questions. These could be addressed if clones that have once demonstrated a specific phenotype in a later phase of cell progression can be isolated from the initial cell population. The new concept of “retrospective clone isolation” has recently emerged to tackle the above-raised questions. A barcoded cell population is first propagated in such a system, and its subpopulation is subjected to a given assay. After identifying a barcoded clone of interest, the same clone (or its close relative) is isolated in a barcode-specific manner from the initial or any other subpopulation stored during the experiment. The isolated live clone can then be subjected to any following experiments, including omics measurements and reconstitution of a synthetic cell population with the isolates. We have recently established a high-performance retrospective isolation technology CloneSelect using CRISPR base editing (bioRxiv 2022). We have demonstrated that CloneSelect is applicable in human cancer cell lines, human pluripotent stem cells, mouse stem cells, yeast cells, and E. coli cells.

We have also been contributing to the development of laboratory automation and data analysis automation platforms. We have previously established humanoid robots to automate various molecular and cellular biology experiments and proposed the vision of robotic cloud biology (Nature Biotechnology 2017). More recently, we have established a new semantic framework to efficiently share reproducible DNA materials and construction protocols within the broad life sciences community (Nature Communications 2022). Furthermore, we have developed a universal tool to interpret and translate high-throughput sequencing read structures, thereby accelerating the development of new sequencing-based assays (Science Advances 2023). In the genomics space, we have established a new theoretical framework to scalably measure the spatial genomics of large tissues without the need for a microscopy system (Cell Systems 2023).

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