Our improved CRISPR-Cas9-based photoactivatable transcription systems, CPTS2.0 and Split-CPTS2.0, enable high blue-light-inducible activation of endogenous target genes in various human cell lines. We achieved reversible activation of target genes with CPTS2.0 and induced neuronal differentiation in induced pluripotent stem cells (iPSCs) by upregulating NEUROD1 with Split-CPTS2.0.

Genome engineering in plants is highly dependent on the availability of effective molecular techniques. Despite vast quantities of research, genome engineering in plants is still limited in terms of gene delivery, which requires the use of infectious bacteria or harsh conditions owing to the difficulty delivering biomaterial into plant cells through the cell wall. Here, we describe a method that uses electroporation-mediated protein delivery into cultured Arabidopsis thaliana cells possessing an intact cell wall, and demonstrate Cre-mediated site-specific recombination. By optimizing conditions for the electric pulse, protein concentration, and electroporation buffer, we were able to achieve efficient and less-toxic protein delivery into Arabidopsis thaliana cells with 83% efficiency despite the cell wall. To the best of our knowledge, this is the first report demonstrating the electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering in plant cells possessing an intact cell wall.

Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to their multilineage differentiation and self-renewal properties. Understanding the in vivo differentiation process is necessary for clinical applications including cell therapy and transplantation. This remains challenging owing to the lack of induction methods that imitate the natural programming process. Endogenous gene regulation of tissue-specific transcription factors is therefore desirable. In the present study, we demonstrated endogenous activation of adipogenic genes through the dCas9-based transcription system and achieved efficient induction of different types of adipocyte-like cells from MSCs. Interestingly, the MSCs converted via single-gene activation exhibited morphological and molecular properties of white adipocytes, while beige adipocyte-like cells were induced via multiplex gene activation of three specific transcription factors. These results reveal that the fate of MSCs can be effectively manipulated by direct activation of specific endogenous gene expression using a dCas9-based activator with reduced exogenous additives.

Wound-healing factors secreted from mesenchymal stem cells (MSCs) modulate the immune response and facilitate proliferation of neighboring cells. Although in vitro three-dimensional (3D) culture techniques have improved the therapeutic potential of MSCs, no studies have focused on the effects of cell aggregation alone. In this study, the effect of cell aggregation on the up-regulation of wound-healing proteins secretions was investigated by constructing small spheroids of human adipose-derived stem cells (hADSCs) on a micropatterned surface. These spheroids were mostly unaffected by the secondary effects of cell aggregation, such as hypoxia, low-nutrient supply, and metabolic waste accumulation. Small spheroids of hADSCs, which were of 100 lm in diameter, were successfully constructed using micropatterned surface. Expression of the wound-healing-related factors, VEGF-A and IL-8, was markedly enhanced at the gene and protein levels, whereas the enhancement of VEGF-A expression was transient and IL-8 enhancement was maintained for a long time.

Bioluminescent detection has become a powerful method that is used extensively in numerous areas in life science research. Given that fluorescence detection in plant cells is difficult owing to the autofluorescence of chlorophyll, the use of a luciferin-luciferase system should be effective in plant biology. However, the suitable optical window for a luminescence system in plants remains unexplored. In this study, we sought to determine the optical window and optimal luciferase reporter system for terrestrial plant analyses using Arabidopsis thaliana as a model organism. We compared six different luciferase systems and found the green enhanced Nano-lantern (GeNL)-furimazine combination to be the optimal luciferase reporter. Spectral measurements of GeNL-furimazine showed that its luminescence peak falls within the range of optical transparency for chlorophyll and, therefore, enables greater penetration through a layer of cultured A. thaliana cells. Moreover, A. thaliana plants expressing GeNL with furimazine emitted strong luminescence, which could be detected even with the naked eye. Thus, the GeNL-furimazine combination should facilitate biological analyses of genes and cellular functions in A. thaliana and all other terrestrial plants.

Lineage commitment of stem cells is mainly regulated by their microenvironments, which comprise soluble growth factors, extracellular matrix, mechanical forces, and cell density. Although numerous studies have investigated stem cell response to these factors in two-dimensional (2D) culture, little is known about that in 3D culture. Here, we studied effects of 3D cell accumulation levels on the differentiation behavior of mesenchymal stem cells (MSCs) by using a micropatterned surface. After induction of 3D-cultured MSCs on the surface, their osteogenic differentiation was significantly promoted, while adipogenic differentiation was not. This differentiation behavior of densely packed MSCs in 3D culture is unlike that in 2D culture. Moreover, to determine the contributing factor of this commitment, the relationship between 3D cell accumulation levels and their differentiation potential was studied before differentiation induction. A series of MSCs with varied 3D accumulation levels were constructed on the micropatterned surface, where the accumulated MSCs were not in hypoxic environment. Interestingly, with increasing 3D accumulation levels, MSCs enhanced their osteogenic potential but repressed adipogenic potential in the gene expression level. These results suggest that preconditioned 3D microenvironments with high cell accumulation levels promote osteogenic differentiation of MSCs and their accumulation levels help in regulating MSC differentiation.

RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable. Here, we focused on Broccoli, a superior variant of Spinach, which is a well-known fluorescent RNA aptamer that binds to DFHBI-1T and emits green fluorescence. We experimentally characterized Broccoli and predicted that it forms a G-quadruplex–based DFHBI-1T recognition region sandwiched between two stems. Based on this, we designed a Broccoli-based RNA detection probe (BRD probe) composed of a sequence of destabilized Broccoli fused with complementary sequences against target RNA. The resulting probe with its target RNA formed a stable three-way junction, named the MT2 three-way junction, which contributed to efficient refolding of the Broccoli structure and allowed for programmable RNA detection with high signal-to-noise ratio and sensitivity. Interestingly, the MT2 three-way junction also could be applied to probe construction of a truncated form of Spinach (Baby Spinach). The BRD and Baby Spinach–based RNA detection probes (BSRD probe) exhibited up to 48- and 140-fold fluorescence enhancements in the presence of their target RNAs and detected small amounts of target RNA that were as low as 160 and 5 nM, respectively. Thus, we experimentally characterized the higher order structure of Broccoli and developed structure-switching aptamer probes for highly sensitive, programmable, RNA detection using an MT2 three-way junction.

CRISPR-Cas9 technology has been at the forefront of the field of biology. The Streptococcus pyogenes (SpyCas9) protein forms a complex with guide RNA and can recognize and cleave double-stranded DNA through hybridization based on 20 base pairings. SpyCas9 has two nuclease domains, HNH and RuvC, each of which cuts each DNA strand, and both contain critical histidine residues. Although previously reported crystal structures provide useful geometric information, the extent to which these residues functionally contribute to catalysis is unknown. Here, we mutated histidine residues on HNH and RuvC domains to alanine or glycine and attempted to rescue the enzymatic activity by adding the imidazole molecule, using an in vitro DNA cleavage assay. H840A and H840G exhibited rescued enzymatic activity on the HNH domain following imidazole addition, suggesting that H840 acts as a general base. We also tested various chemicals and found that the pK a of imidazole derivatives, and not their molecular shape, correlated with the rescue effect. In contrast, both H983A and H983G on the RuvC domain did not exhibit a rescue effect following imidazole addition. Our chemical rescue approach will provide crucial insight into understanding Cas9 catalysis, complementing structural analyses.