Efficient genome editing by controlled release of Cas9 ribonucleoprotein in plant cytosol using polymer-modified microneedle array.

Direct delivery of genome-editing proteins into plant tissues could be useful in obtaining DNA-free genome-edited crops obviating the need for backcrossing to remove vector-derived DNA from the host genome as in the case of genetically modified organisms generated using DNA vector. Previously, we successfully delivered Cas9 ribonucleoprotein (RNP) into plant tissue by inserting microneedle array (MNA) physisorbed with Cas9 RNPs. Here, to enhance protein delivery and improve genome-editing efficiency, we introduced a bioactive polymer DMA/HPA/NHS modification to the MNA, which allowed strong bonding between the proteins and MNA. Compared with other modifying agents, this MNA modification resulted in better release of immobilized protein in a plant cytosol-mimicking environment. The delivery of Cas9 RNPs in Arabidopsis thaliana reporter plants was improved from 4 out of 17 leaf tissues when using unmodified MNAs to 9 out of 17 when using the polymer-modified MNAs. Further improvements in delivery efficiency can be envisaged by optimizing the polymer modification conditions, which could have significant implications for the development of more effective plant genome editing techniques.

Asymmetric Roles of Two Histidine Residues in Streptococcus pyogenes Cas9 Catalytic Domains upon Chemical Rescue.

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 pKa 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.

Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation.

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-based photoactivatable transcription systems to induce neuronal differentiation.

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.

Small spheroids of adipose-derived stem cells with time-dependent enhancement of IL-8 and VEGF-A secretion.

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 µm 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.

A Method for Electroporation of Cre Recombinase Protein into Intact Nicotiana tabacum Cells.

The Cre/lox recombination system has become a powerful technology for gene function analysis in a broad spectrum of cell types and organisms. In our previous report, Cre protein had been successfully delivered into intact Arabidopsis thaliana cells using electroporation. To expand the feasibility of the method of protein electroporation to other plant cells, here we attempt the protein electroporation into tobacco-derived BY-2 cells, one of the most frequently used plant cell lines for industrial production. In this study, we successfully deliver Cre protein into BY-2 cells with intact cell walls by electroporation with low toxicity. Targeted loxP sequences in the BY-2 genome are recombined significantly. These results provide useful information for genome engineering in diverse plant cells possessing various types of cell walls.

Microneedle Array-Assisted, Direct Delivery of Genome-Editing Proteins Into Plant Tissue.

Genome editing in plants employing recombinant DNA often results in the incorporation of foreign DNA into the host genome. The direct delivery of genome-editing proteins into plant tissues is desired to prevent undesirable genetic alterations. However, in most currently available methods, the point of entry of the genome-editing proteins cannot be controlled and time-consuming processes are required to select the successfully transferred samples. To overcome these limitations, we considered a novel microneedle array (MNA)-based delivery system, in which the needles are horizontally aligned from the substrate surface, giving it a comb-like configuration. We aimed to deliver genome-editing proteins directly into the inner layers of leaf tissues; palisade, the spongy and subepidermal L2 layers of the shoot apical meristem (SAM) which include cells that can differentiate into germlines. The array with needles 2 µm wide and 60 µm long was effective in inserting into Arabidopsis thaliana leaves and Glycine max (L.) Merr. (soybeans) SAM without the needles buckling or breaking. The setup was initially tested for the delivery of Cre recombinase into the leaves of the reporter plant A. thaliana by quantifying the GUS (ß-glucuronidase) expression that occurred by the recombination of the loxP sites. We observed GUS expression at every insertion. Additionally, direct delivery of Cas9 ribonucleoprotein (RNP) targeting the PDS11/18 gene in soybean SAM showed an 11 bp deletion in the Cas9 RNP target site. Therefore, this method effectively delivered genome-editing proteins into plant tissues with precise control over the point of entry.

Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system.

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.

A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall.

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.

Control of Adipogenic Differentiation in Mesenchymal Stem Cells via Endogenous Gene Activation Using CRISPR-Cas9.

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.

Osteogenic Lineage Commitment of Adipose-Derived Stem Cells Is Predetermined by Three-Dimensional Cell Accumulation on Micropatterned Surface.

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.