隨著 CRISPR/Cas9 基因編輯技術的演進，如何精準且穩定地將大型外源片段導入植物基因組，已成為植物分子農業領域亟待解決的核心課題。傳統的同源定向修復（Homology-Directed Repair， HDR）在植物體細胞中的發生頻率極低，且對於超過 2 kb 的基因片段承載能力有限。為克服此瓶頸，本研究轉向利用植物中最為活躍的雙股斷裂修復機制——非同源末端連接（NHEJ）。透過在供體質體上設計與基因組標靶相同的 sgRNA 識別位點，使其在細胞內經 Cas9 剪切後釋放出線性供體片段，並透過 NHEJ 介導的縫合機制，提升長度達 2.2 kb 之 rcDEL1−IgGFC 融合基因在水稻基因組中的定點整合效率。在整合位點的選取上，本研究選定具糖飢餓誘導特性的水稻 αAmy3 基因第一內含子作為標的，利用其非編碼特性降低 NHEJ 修復過程中末端鹼基隨機增減（Indels）對蛋白質開放閱讀框的影響。為確保轉錄產物的精確性與連續性，本研究於外源基因前端配置剪接接受子（Splice acceptor， SA），使插入片段可被宿主剪接體辨識並視為內含子的一部分進行處理，形成同框（in-frame）的融合 mRNA，確保 DEL-1 蛋白在轉譯後能順利進入內質網與高基氏體進行折疊與加工。此分泌至培養基的生產模式對DEL-1 類複雜醣蛋白具有重要應用價值，不僅可簡化下游純化製程並降低成本，亦能避免蛋白於胞內累積所造成的內質網壓力與代謝負擔，維持水稻懸浮細胞在誘導期間的生長活性。此外，培養基環境亦有助於維持蛋白結構完整並降低蛋白酶降解風險。本研究最終從 108 株轉殖組織中篩選出 4 株具高精確敲入特性的細胞株，顯示該分泌平台在處理大型治療性蛋白基因時具備良好效率與遺傳穩定性，並為未來規模化生產低成本、具生物安全性之牙周再生藥物奠定實驗基礎。;To address the limitations of Homology-Directed Repair (HDR) in integrating large gene fragments in plants， this study developed an NHEJ-mediated "decoy and stitch" strategy to precisely knock in a 2.2 kb rcDEL1−IgGFC fusion gene into the first intron of the rice αAmy3 gene. By designing sgRNA recognition sites on the donor plasmid to release linear fragments within the cell， we significantly enhanced the targeted integration efficiency. A critical component of this strategy is the inclusion of a splice acceptor (SA) at the 5′ end of the donor， which ensures the large heterologous insert is accurately processed by the host spliceosome， resulting in mature， in-frame fusion mRNA between the native signal peptide and the target protein. This secretion-based expression platform offers several strategic advantages for the production of complex glycoproteins like DEL-1. First， the direct release of the target protein into the culture medium simplifies downstream processing by eliminating the need for cell lysis and the subsequent removal of host cell contaminants. Second， the secretion mechanism effectively alleviates endoplasmic reticulum (ER) stress and metabolic burden caused by intracellular protein accumulation， maintaining robust cell growth kinetics. Furthermore， the extracellular microenvironment helps preserve the structural integrity of DEL-1 against intracellular protease degradation. Ultimately， 4 high-precision knock-in lines were successfully identified from 108 transgenic tissues， demonstrating the platform′s efficiency and genetic stability. This system provides a solid foundation for the low-cost， large-scale production of therapeutic proteins for periodontal regeneration.
