A mortise-tenon joint system facilitates precise targeted DNA insertion and replacement in rice.

Precise and scarless DNA insertion and replacement represent two of the major challenges in plant genome editing. Numerous tools have been developed, including homology-directed repair-dependent tools and prime-editing-mediated systems. However, none has fully resolved these challenges. In this study, we develop the "mortise-tenon joint system" (MT), a novel strategy that enables precise and efficient targeted DNA insertion and replacement. By leveraging the APOBEC-Cas9-uracil DNA glycosylase/(apurinic or apyrimidinic site) lyase within our previously reported APOBEC-Cas9 fusion-induced deletion system, which performs single-strand cleavage on the non-target strand and double-strand cleavage on the target strand, we generated a unique "mortise" structure, consisting of double-strand breaks with single or double non-complementary 5' overhangs. We further designed "tenons," double-stranded DNA donors containing 5' sticky ends precisely matching 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% across seven tested targets using 21-85 bp donor inserts in rice. If long DNA donors with sticky ends complementary to the mortise structure can be generated, the MT system may enable highly precise targeted insertion and replacement of large DNA fragments.