cRiSpR/cas-mediated genome editing is a powerful tool for generating genetically mutated cells and organisms. Linearisation of donor cassettes with this system has been shown to facilitate both transgene donor insertion and targeted knock-in. Here, we developed a donor plasmid that we name pCriMGET (plasmid of synthetic cRiSpR coded RnA target sequence-equipped donor plasmidmediated gene targeting), in which an off-target free synthetic CRISPR coded RNA-target sequence (syn-crRNA-TS) is incorporated with a multi-cloning site, where a donor cassette can be inserted. With co-expression of Cas9 and the syn-crRNA-TS guide RNA (gRNA), pCriMGET provides a linearised donor cassette in vivo, thereby promoting the transgene donor insertion and targeted knock-in. When co-injected with Cas9 protein and gRNA into murine zygotes, pCriMGET yielded around 20% transgene insertion in embryos. This method also achieved more than 25% in-frame knock-in at the mouse Tbx3 gene locus without predicted insertion-deletion mutations using a transgene donor with 400-bp homology arms. pCriMGET is therefore useful as a versatile CRISPR/Cas9-cleavable donor plasmid for efficient integration and targeted knock-in of exogenous DNA in mice. The generation of genetically mutated animals is essential for studies of gene function and pathological analysis in vivo 1,2. The genome editing methods using site-specific nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like nucleases (TALENs) and RNA-guided nucleases, have made it possible to integrate exogenous DNA into targeted genomic loci 3. In particular, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has been widely used because of its simple procedures and high versatility in targeting genomic loci. Using a guide RNA (gRNA) for a target sequence, CRISPR/Cas induces double-strand breaks (DSBs) in the target genomic locus, at which exogenous DNA can be integrated via DNA repair pathways. Because the gene targeting efficiency of the conventional method based on the homologous recombination (HR) pathway is low 4,5 , substantial efforts have been made to develop efficient and precise targeted knock-in methods. The strategies based on the repair pathways of nonhomologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) have achieved targeted transgene integration in zebrafish and mice with no or short homology arms on both sides of the donor cassette 6-13. However, these methods often cause undesired insertion-deletion (indel) mutations at the DSB sites 6-13. Other groups reported the Easi-CRISPR targeting method for the generation of knock-in mice using long single-stranded DNA (ssDNA) as a donor template 14,15. However, this method has a limitation in terms of the donor size (< 2 kb) and is prone to cause rearranged alleles including indels 16. The Tild-CRISPR method, in which PCR-amplified or in vitro cleaved linearised double-stranded DNA is using as a donor, exhibits high knock-in efficiency in mice 17. ...

Stem cell (SC) proliferation and differentiation organize tissue homeostasis. However, how SCs regulate coordinate tissue scaling in dynamic organs remain unknown. Here, we delineate SC regulations in dynamic skin. We found that interfollicular epidermal SCs (IFESCs) shape basal epidermal proliferating clusters (EPCs) in expanding abdominal epidermis of pregnant mice and proliferating plantar epidermis. EPCs consist of IFESC-derived Tbx3+–basal cells (Tbx3+-BCs) and their neighboring cells where Adam8–extracellular signal–regulated kinase signaling is activated. Clonal lineage tracing revealed that Tbx3+-BC clones emerge in the abdominal epidermis during pregnancy, followed by differentiation after parturition. In the plantar epidermis, Tbx3+-BCs are sustained as long-lived SCs to maintain EPCs invariably. We showed that Tbx3+-BCs are vasculature-dependent IFESCs and identified mechanical stretch as an external cue for the vasculature-driven EPC formation. Our results uncover vasculature-mediated IFESC regulations, which explain how the epidermis adjusts its size in orchestration with dermal constituents in dynamic skin.

The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.

Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.Insc was first identified as a novel neural precursor gene in Drosophila (1). Insc protein expression has been detected in embryonic areas where cell shape changes or movement occurs (i.e. neuroectoderm, midgut primordium, and muscle precursors) (1). More precise roles have emerged for Insc protein activity based on studies using neuroblasts, stem cells found in the central nervous system of Drosophila, which undergo asymmetric cell division (2-5). In neuroblasts, Insc localizes to the apical cell cortex by directly associating with bazooka-Par6-aPKC cell polarity protein complexes, whereas cell fate determinants, such as miranda (Mira), prospero (Pros), brain tumor (Brat), and Numb, localize to the basal cortex (6 -20). Alignment of the mitotic spindle along the apical-basal polarity axis drives asymmetric cell division to produce one self-renewing neuroblast and one ganglion mother cell fated for neural differentiation by asymmetrical inheritance of cell fate determinants (6,10,11,13,21,22). Insc plays a critical role in apical-basal spindle positioning by connecting spindle capturing machineries, consisting of partner of inscuteable (Pins) and mushroom body defect (Mud), with apical bazooka-Par6-aPKC cell polarity complexes (23-26).Similar molecular machineries are conserved in neural progenitors (27, 28) and skin basal cells of mice (29 -31), whereby mouse INSC (the mouse homologue of mammalian inscuteable) regulates spindle orientation and cell fate determination together with Par-3 (vertebrate homolog of Bazooka) and LGN (vertebrate counterpart of Pins) (27-31). Previous reports show that ectopic expression of mouse INSC promotes apical-basal spindle positioning in neuronal progenitors and keratin...