原 弘真

CRISPR/Cas9-mediated genome editing has expanded the possibilities for precise gene modifications; however, the efficiency of targeted insertion remains suboptimal. In this study, we describe a triple-reporter system in mouse embryonic stem cells that simultaneously tracks double-strand break (DSB) induction, homology-directed repair (knock-in), and end-joining-mediated targeted insertion (EJ-TI). Using both plasmid and adeno-associated virus (AAV) donor vectors, our results demonstrate that ataxia telangiectasia and Rad3-related kinase (ATR) activity is essential for knock-in regardless of the donor type, whereas ataxia telangiectasia mutated (ATM) inhibition exhibits a donor-dependent role. In cells receiving circular plasmid donors, ATM inhibition with AZD1390 markedly reduced the knock-in and EJ-TI efficiencies, consistent with its canonical role in DSB repair. In contrast, with linear AAV donors, ATM inhibition enhanced the knock-in efficiency by suppressing the overactivation of the ATM-p53-caspase 3 apoptotic pathway and partially suppressing classical non-homologous end-joining. These findings highlight the critical influence of donor DNA configuration on DNA damage response signaling and provide a strategy for optimizing genome editing efficiency by selectively modulating the ATM pathways, an approach that may have significant implications for gene therapy, cell engineering, and other applications.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.

Knock-in therapy, in which an insertion site can be controlled, would be more suitable for the treatment of genetic blood disorders as compared to conventional gene therapy with lentivirus vectors that introduce genes into the genome randomly. Recent advancements in genome editing technology have substantially improved the knock-in efficiency, making it a reality. We present the details of a virus-free CRISPR/Cas9-based genome editing method for bona fide mouse hematopoietic stem cells.