水稻（Oryza sativa）懸浮細胞培養系統因其具有可擴展性、高度封閉性以及能進行複雜的轉譯後修飾等優勢，被視為具有潛力的重組蛋白質生產平台。本研究旨在透過兩種獨立策略—轉錄調控與CRISPR/Cas9基因組工程技術，提升由糖飢餓誘導的α-澱粉酶（α-Amylase）啟動子驅動的重組蛋白質表現。
第一種策略運用RNA干擾（RNAi）技術抑制OsMYBS2基因的表達。OsMYBS2是一種MYB相關的轉錄因子，作為抑制因子與激活因子OsMYBS1競爭結合於αAmy3啟動子的TA盒上。為驗證此方法，本研究選擇利用經水稻密碼子優化的重組小鼠粒細胞-巨噬細胞集落刺激因子（rmGM-CSF）作為模式蛋白。結果顯示rmGM-CSF蛋白產量達69.8 µg/mL，較對照組提升2.5倍。此外，在純合F5世代中，rmGM-CSF產量更進一步提高至118.8 µg/mL，相較對照組提升5.1倍。
另一種策略採用CRISPR/Cas9介導的基因敲入技術，將經過水稻密碼子優化的SARS-CoV-2刺突蛋白受體結合域（rcRBD），以單一供體載體插入至αAmy3或αAmy8基因第一內含子位置。rcRBD的表達受各基因座內源性啟動子及信號肽調控。當rcRBD整合於αAmy3基因座時，蛋白質可在細胞內與培養液中累積；而整合於αAmy8基因座時，則專一性產生分泌型蛋白。尤其是透過αAmy8啟動子所達之最高分泌蛋白量為20.7 mg/L，超越既有植物表達系統的產量水平。
綜合上述結果，本研究證實，無論是針對 αAmy3 或 αAmy8 基因，採用轉錄重編程或CRISPR/Cas9單一供體導向的位點特異性基因整合，皆為有效且具應用潛力的策略，可顯著提升水稻懸浮細胞的重組蛋白產量。

Rice (Oryza sativa) suspension cell cultures are a promising platform for recombinant protein production due to their scalability, containment, and ability to undertake complicated post-translational modifications. This study aimed to enhance the expression of recombinant proteins driven by the sugar starvation-inducible α-Amylase promoter through two independent approaches, transcriptional regulation and genome engineering techniques by CRISPR/Cas9.
The first approach involved RNA interference (RNAi) to knock down the expression of the OsMYBS2 gene, a MYB-related transcription factor known as a repressor that competes with the activator OsMYBS1 at the TA box of the αAmy3 promoter. To assess this method, a recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) optimized for rice codon usage was expressed under the control of the αAmy3 promoter. Silencing OsMYBS2 significantly increased both transcript levels and protein yield of αAmy3 and rmGM-CSF, achieving the yield of rmGM-CSF protein reached to 69.8 µg/mL, which corresponds to a 2.5-fold enhancement relative to non-silenced controls. Moreover, in cultured cells derived from homozygous F5 seeds, rmGM-CSF production further increased to 118.8 µg/mL, representing a 5.1-fold improvement compared to the control line.
The second approach utilized a CRISPR/Cas9-mediated knock-in strategy to insert a rice codon-optimized receptor-binding domain (rcRBD) of the SARS-CoV-2 spike protein into the first intron of either the αAmy3 or αAmy8 genes, using a single donor cassette. Expression was driven by the endogenous promoters and signal peptides corresponding to each locus. When integrated at the αAmy3 locus, rcRBD protein accumulated both intracellularly and extracellularly, whereas insertion at the αAmy8 locus resulted exclusively in secreted protein. Notably, the highest secretion level, achieved via the αAmy8 promoter, reached 20.7 mg/L, surpassing the production efficiency reported in previous plant-based expression systems.
Together, these results demonstrate that transcriptional reprogramming and CRISPR/Cas9-directed locus-specific gene integration by using single donor for targeting either αAmy3 or αAmy8 genes are two distinct and potent strategies for enhancing recombinant protein production in rice suspension cultures.

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