SpCas9-HF1 enhances accuracy of cell cycle-dependent genome editing by increasing HDR efficiency, and by reducing off-target effects and indel rates.

In genome editing, it is important to avoid off-target mutations so as to reduce unexpected side effects, especially for therapeutic applications. Recently, several high-fidelity versions of SpCas9 have been developed to reduce off-target mutations. In addition to reducing off-target effects, highly efficient intended target gene correction is also essential to rescue protein functions that have been disrupted by single nucleotide polymorphisms. Homology-directed repair (HDR) corrects genes precisely using a DNA template. Our recent development of cell cycle-dependent genome editing has shown that regulation of Cas9 activation with an anti-CRISPR-Cdt1 fusion protein increases HDR efficiency and reduces off-target effects. In this study, to apply high-fidelity SpCas9 variants to cell cycle-dependent genome editing, we evaluated anti-CRISPR inhibition of high-fidelity SpCas9s. In addition, HDR efficiency of high-fidelity SpCas9s was addressed, identifying eSpCas9, SpCas9-HF1, and LZ3 Cas9 as promising candidates. Although eSpCas9 and LZ3 Cas9 showed decreased HDR efficiency in cell cycle-dependent genome editing, SpCas9-HF1 successfully achieved increased HDR efficiency and few off-target effects when co-expressed with an AcrIIA4-Cdt1 fusion.

Formation of the brain by stem cell divisions of large neuroblasts in Oikopleura dioica, a simple chordate.

Stem cell division contributes to the generation of various cell types during animal development, especially a diverse pool of neural cells in the nervous system. One example is reiterated unequal stem cell divisions, in which a large stem cell undergoes a series of oriented unequal divisions to produce a chain of small daughter cells that differentiate. We show that reiterated unequal stem cell divisions are involved in the formation of the brain in simple chordate appendicularians (larvaceans). Two large neuroblasts in the anterior and middle of the brain-forming region of hatched larvae were observed. They produced at least 30 neural cells out of 96 total brain cells before completion of brain formation at 10 hours after fertilization by reiterated unequal stem cell divisions. The daughter cells of the anterior neuroblast were postmitotic, and the number was at least 19. The neuroblast produced small daughter neural cells posteriorly every 20 min. The neural cells first moved toward the dorsal side, turned in the anterior direction, aligned in a single line according to their birth order, and showed collective movement to accumulate in the anterior part of the brain. The anterior neuroblast originated from the right-anterior blastomeres of the eight-cell embryos and the right a222 blastomere of the 64-cell embryo. The posterior neuroblast also showed reiterated unequal stem cell divisions, and generated at least 11 neural cells. Sequential unequal stem cell divisions without stem cell growth have been observed in protostomes, such as insects and annelids. The results provide the first examples of this kind of stem cell division during brain formation in non-vertebrate deuterostomes.

Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy.

Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.

The past, present, and future of artificial zinc finger proteins: design strategies and chemical and biological applications.

Zinc finger proteins are abundant in the human proteome and are responsible for a variety of functions. The domains that constitute zinc finger proteins are compact spherical structures, each comprising approximately 30 amino acid residues, but they also have precise molecular factor functions: zinc binding and DNA recognition. Due to the biological importance of zinc finger proteins and their unique structural and functional properties, many artificial zinc finger proteins have been created and are expected to improve their functions and biological applications. In this study, we review previous studies on the redesign and application of artificial zinc finger proteins, focusing on the experimental results obtained by our research group. In addition, we systematically review various design strategies used to construct artificial zinc finger proteins and discuss in detail their potential biological applications, including gene editing. This review will provide relevant information to researchers involved or interested in the field of artificial zinc finger proteins as a potential new treatment for various diseases.

An ImageJ-based tool for three-dimensional registration between different types of microscopic images.

Three-dimensional (3D) registration (i.e., alignment) between two microscopic images is very helpful to study tissues that do not adhere to substrates, such as mouse embryos and organoids, which are often 3D rotated during imaging. However, there is no 3D registration tool easily accessible for experimental biologists. Here we developed an ImageJ-based tool which allows for 3D registration accompanied with both quantitative evaluation of the accuracy and reconstruction of 3D rotated images. In this tool, several landmarks are manually provided in two images to be aligned, and 3D rotation is computed so that the distances between the paired landmarks from the two images are minimized. By simultaneously providing multiple points (e.g., all nuclei in the regions of interest) other than the landmarks in the two images, the correspondence of each point between the two images, i.e., to which nucleus in one image a certain nucleus in another image corresponds, is quantitatively explored. Furthermore, 3D rotation is applied to one of the two images, resulting in reconstruction of 3D rotated images. We demonstrated that this tool successfully achieved 3D registration and reconstruction of images in mouse pre- and post-implantation embryos, where one image was obtained during live imaging and another image was obtained from fixed embryos after live imaging. This approach provides a versatile tool applicable for various tissues and species.

Importance of two-dimensional cation clusters induced by protein folding in intrinsic intracellular membrane permeability.

We investigated the cell penetration of Sp1 zinc finger proteins (Sp1 ZF) and the mechanism via which the total cationic charge and distribution of cationic residues on the protein surface affect intracellular trafficking. Sp1 ZFs showed intrinsic cell membrane permeability. The intracellular transfer of Sp1 ZFs other than 1F3 was dependent on the total cationic charge. Investigation of the effect of cationic residue distribution on intracellular membrane permeability revealed that the cellular uptake of unfolded Zn2+-non-coordinating Ala mutants was lower than that of the wild type. Therefore, the total cationic charge and distribution of cationic residues on the protein played crucial roles in intracellular translocation. Mutational studies revealed that the two-dimensional cation cluster on the protein surface significantly improved their cellular uptake. This study will contribute to the design of artificial cargoes that can efficiently transport target substances into cells.

Repetitive short-pulsed illumination efficiently activates photoactivatable-Cre as continuous illumination in embryonic stem cells and pre-implantation embryos of transgenic mouse.

The Cre-loxP system has been widely used for specific DNA recombination which induces gene inactivation or expression. Recently, photoactivatable-Cre (PA-Cre) proteins have been developed as a tool for spatiotemporal control of the enzymatic activity of Cre recombinase. Here, we generated transgenic mice bearing a PA-Cre gene and systematically investigated the conditions of photoactivation for the PA-Cre in embryonic stem cells (ESCs) derived from the transgenic mice and in a simple mathematical model. Cre-mediated DNA recombination was induced in 16% of the PA-Cre ESCs by 6 hr continuous illumination. We show that repetitive pulsed illumination efficiently induced DNA recombination with low light energy as efficient as continuous illumination in the ESCs (96 ± 15% of continuous illumination when pulse cycle was 2 s), which was also supported by a minimal mathematical model. DNA recombination by the PA-Cre was also successfully induced in the transgenic mouse pre-implantation embryos under the developed conditions. These results suggest that strategies based on repetitive pulsed illumination are efficient for the activation of photoactivatable Cre and, possibly other photo-switchable proteins.

A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left-right patterning.

Larvaceans are chordates with a tadpole-like morphology. In contrast to most chordates of which early embryonic morphology is bilaterally symmetric and the left-right (L-R) axis is specified by the Nodal pathway later on, invariant L-R asymmetry emerges in four-cell embryos of larvaceans. The asymmetric cell arrangements exist through development of the tailbud. The tail thus twists 90° in a counterclockwise direction relative to the trunk, and the tail nerve cord localizes on the left side. Here, we demonstrate that larvacean embryos have nonconventional L-R asymmetries: 1) L- and R-cells of the two-cell embryo had remarkably asymmetric cell fates; 2) Ca2+ oscillation occurred through embryogenesis; 3) Nodal, an evolutionarily conserved left-determining gene, was absent in the genome; and 4) bone morphogenetic protein gene (Bmp) homolog Bmp.a showed right-sided expression in the tailbud and larvae. We also showed that Ca2+ oscillation is required for Bmp.a expression, and that BMP signaling suppresses ectopic expression of neural genes. These results indicate that there is a chordate species lacking Nodal that utilizes Ca2+ oscillation and Bmp.a for embryonic L-R patterning. The right-side Bmp.a expression may have arisen via cooption of conventional BMP signaling in order to restrict neural gene expression on the left side.

Patterning and morphogenesis of the intricate but stereotyped oikoplastic epidermis of the appendicularian, Oikopleura dioica.

Mechanisms for morphogenetic processes that generate complex patterns in a reproducible manner remain elusive. Live imaging provides a powerful tool to record cell behaviors. The appendicularian, Oikopleura dioica, is a planktonic tunicate that has a rapid developmental speed, small number of cells (less than 3500 cells in a juvenile), and a transparent body. The trunk epidermis, called the oikoplastic epithelium (OE), has elaborate cellular arrangements showing a complex pattern to secrete so-called "house" made of extracellular components. The OE is characterized by invariant number, size, and shape of the monolayer epithelial cells. Pattern formation is achieved during 5h of larval development without growth of the body, making this a suitable system for live imaging of a two-dimensional (2D) sheet. First, we subdivided the OE and defined several domains by cellular resolution, and systematically gave names to the constituent cells, since there is no variation among individuals. Time-lapse imaging of the epidermal cells revealed region-specific pattern formation processes. Each identified domain served as a compartment into which distribution of descendant cells of founder cells is restricted. Regulation of orientation, timing, and the number of rounds of cell divisions, but not cell death and migration, was a critical mechanism for determination of final cell arrangement and size. In addition, displacement of epithelial sheet plates was observed in the Eisen domain. Stem-cell-like cell divisions, whereby large mother stem cells generate a chain of small daughter cells, were involved in formation of the Nasse region and ventral sensory organ. These are the first examples of this kind of stem-cell-like cell division in deuterostomes. Furthermore, labeling of the left or right blastomere of the two-cell-stage embryo, which roughly gives rise to the left or right side of the body, respectively, revealed that the boundary of the descendant cells does not match with the midline of the trunk epidermis. Left and right descendants largely invade into the opposite side in an invariant way, suggesting the possibility that specification of the OE cell identities may occur later in development, most probably around hatching, and depending on cell position in the OE epithelial sheet. These detailed descriptions of OE patterning processes provide basic and essential information to analyze further cell behaviors in the generation of elaborate and intricate but stereotyped 2D cellular patterns in this advantageous model system for developmental and cell biological studies in chordates.

Maternal and zygotic transcriptomes in the appendicularian, Oikopleura dioica: novel protein-encoding genes, intra-species sequence variations, and trans-spliced RNA leader.

RNA sequencing analysis was carried out to characterize egg and larval transcriptomes in the appendicularian, Oikopleura dioica, a planktonic chordate, which is characterized by rapid development and short life cycle of 5 days, using a Japanese population of the organism. De novo transcriptome assembly matched with 16,423 proteins corresponding to 95.4% of the protein-encoding genes deposited in the OikoBase, the genome database of the Norwegian population. Nucleotide and amino acid sequence identities between the Japanese and Norwegian O. dioica were estimated to be around 91.0 and 94.8%, respectively. We discovered 175 novel protein-encoding genes: 144 unigenes were common to both the Japanese and Norwegian populations, whereas 31 unigenes were not found in the OikoBase genome reference. Among the total 12,311 unigenes, approximately 63% were detected in egg-stage RNAs, whereas 99% were detected in larval stage RNAs; 3772 genes were up-regulated, and 1336 genes were down-regulated more than four-fold in the larvae. Gene ontology analyses characterized gene activities in these two developmental stages. We found a messenger RNA (mRNA) 5' trans-spliced leader, which was observed in 40.8% of the total unique transcripts. It showed preferential linkage to adenine at the 5' ends of the downstream exons. Trans-splicing was observed more frequently in egg mRNAs compared with larva-specific mRNAs.

Long-distance cell migration during larval development in the appendicularian, Oikopleura dioica.

The appendicularian, Oikopleura dioica, is a planktonic chordate. Its simple and transparent body, invariant cell lineages and short life cycle of 5 days make it a promising model organism for studies of chordate development. Here we describe the cell migration that occurs during development of the O. dioica larva. Using time-lapse imaging facilitated by florescent labeling of cells, three cell populations exhibiting long-distance migration were identified and characterized. These included (i) a multinucleated oral gland precursor that migrates anteriorly within the trunk region and eventually separates into the left and right sides, (ii) endodermal strand cells that are collectively retracted from the tail into the trunk in a tractor movement, and (iii) two subchordal cell precursors that individually migrate out from the trunk to the tip of the tail. The migration of subchordal cell precursors starts when all of the endodermal strand cells enter the trunk, and follows the same path but in a direction opposite to that of the latter. Labeling of these cells with a photoconvertible fluorescent protein, Kaede, demonstrated that the endodermal strand cells and subchordal cell precursors have distinct origins and eventual fates. Surgical removal of the trunk from the tail demonstrated that the endodermal strand cells do not require the trunk for migration, and that the subchordal cell precursors would be attracted by the distal part of the tail. This well-defined, invariant and traceable long-distance cell migration provides a unique experimental system for exploring the mechanisms of versatile cell migration in this simple organism with a chordate body plan.