A Mortise-Tenon Joint System Facilitates Precise Targeted DNA Insertion and Replacement in Rice
    作者: Wenjing Sun, Yaoyao Zhu, Xiuhua Zhang, Xiangbing Meng, Kai Xia, Xin Zhang, Xue Han, Wenbo Pan, Juan Guo, Jiayang Li, Huawei Zhang
    刊物名称: Molecular Plant
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    发布时间: 2025-11-21
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    摘要:
    Precise and scarless DNA insertion and replacement represent two major challenges in plant genome editing. Numerous tools have been employed, including homology-directed repair (HDR)-dependent tools and PE-mediated systems; however, none have fully resolved this challenge. Here, we introduce the “Mortise-Tenon joint system” (MT), a novel strategy that enables precise and efficient targeted insertion and replacement. Leveraging the APOBEC-Cas9-UDG/AP lyase within our previously reported AFID system, which performs single cleavage on the non-target strand and double cleavage on the target strand, we generated the “mortise” structure, a unique type of double-strand breaks (DSBs) with single or double non-complementary 5’-overhangs. Additionally, we designed “tenons”, double-stranded DNA donors containing 5’-sticky ends precisely matched with the 5’-overhangs of the mortises. The end-capture interaction between mortises and tenons facilitates precise targeted insertion and replacement, achieving frequencies of 16.30%-59.47% in seven tested targets with 21-85 bp insertion donors in rice. If long DNA donors with sticky ends complementary to the “mortise” structure can be generated, the MT system holds great promise for enabling the precise target insertion and replacement of large DNA fragments.