担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Efficient targeted integration (TI) of homologous donor DNA is crucial for precise genome editing. Although mismatch repair (MMR) is known to suppress TI as well as homologous recombination (HR) when sequence divergence is present, it remains controversial as to whether MMR affects TI of isogenic donor DNA. In this study, we investigated whether and how the MMR protein Msh2 affects TI of isogenic donor DNA. We found that HR-dependent TI is suppressed by Msh2 only when a homology arm of donor DNA is as short as 1.7 kb. In contrast, single-strand annealing-mediated TI, which is cell cycle-independent and becomes prominent when HR or non-homologous end joining is inactivated, is weakly but constantly affected by Msh2 irrespective of the size of homology arms. Our results reveal a previously unrecognized type of HR suppression by Msh2 and provide implications for precise genome editing using short-arm donor DNA vectors.

DOI： 10.1073/pnas.2508507122

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Homologous recombination (HR) and mismatch repair (MMR) act as guardians of the human genome, and defects in HR or MMR are causative in at least a quarter of all malignant tumors. Although these DNA repair-deficient tumors are eligible for effective targeted therapies, fully reliable diagnostic strategies based on functional assay have yet to be established, potentially limiting safe and proper application of the molecular targeted drugs. Here we show that transient transfection of artificial DNA substrates enables ultrarapid detection of HR and MMR. This finding led us to develop a diagnostic strategy that can determine the cellular HR/MMR status within one day without the need for control cells or tissues. Notably, the accuracy of this method allowed the discovery of a pathogenic RAD51D mutation, which was missed by existing companion diagnostic tests. Our methods, termed HR eye and MMR eye, are applicable to frozen tumor tissues and roughly predict the response to therapy. Overall, the findings presented here could pave the way for accurately assessing malignant tumors with functional defects in HR or MMR, a step forward in accelerating precision medicine.

DOI： 10.1038/s41467-025-59462-2

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bbrc.2024.150977

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.

DOI： 10.1038/s41467-024-49385-9

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Male and female reproductive tracts develop from anterior intermediate mesoderm with similar differentiation processes. The anterior intermediate mesoderm develops into the mesonephros, and the Wolffian duct initiates by epithelialization in the mesonephros. The Müllerian duct invaginates from the coelomic epithelium of the cranial mesonephros for ductal formation and is then regionalized into proximal to caudal female reproductive tracts. In this study, we focused on the epithelialization of the Wolffian duct, initiation of the Müllerian duct, and the regionalization step of the Müllerian ducts as a continuous process. By using intermediate mesodermal cells from mouse pluripotent stem cells, we identified that inhibition of SMAD2/3 signaling might be involved in the differentiation into mesenchymal cells, after which mesonephric cells might be then epithelialized during differentiation of the Wolffian duct. Aggregation of coelomic epithelial cells might be related to initiation of the Müllerian duct. Transcriptomic analysis predicted that consensus sequences of SMAD3/4 were enriched among highly expressed genes in the proximal Müllerian duct. SMAD2/3 signaling to regulate differentiation of the Wolffian duct was continuously activated in the proximal Müllerian duct and was involved in proximal and oviductal regionalization. Therefore, SMAD2/3 signaling may be finely tuned to regulate differentiation from initiation to regionalization steps.

DOI： 10.1002/2211-5463.13729

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記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（指名）  

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