常間回文重複序列叢集干擾系統 (CRISPRi) 是一套新穎的基因干擾技術。此方式首次被利用於微藻 Chlamydomonas reinhardtii CC-400，以調控外源提供的紅色螢光蛋白 (RFP) 基因表達作為展示概念，再以抑制內生磷酸烯醇丙酮酸羧化酶 (PEPC1) 基因作為實際應用。由 RFP 研究分析，CRISPRi 介導的基因調控，在 C. reinhardtii 中的實現效率可達 94%，基於本研究 sgRNA 之靶向位點，仍有 19% 的抑制效率，並且可持續至少 7 代的遺傳穩定性。以 CRISPRi 干擾下調 PEPC1 基因達成控制著進入 TCA 循環的碳通量，由基質互相競爭碳量的分配促進甘油二酯醯基轉移酶 (DGAT) 基因表達並增加油脂合成。實驗結果顯示，所有 CrPEPC1 下調株均具有較低的葉綠素表現水平，在生物質量和油脂累積率則有優越表現，最佳的介導株擁有佔細胞乾重 28.5% 的油脂總量與 34.9 mg/L/day 的油脂產率，相比野生型分別提升了高達 74.4% 和 94.2%。本研究亦調控涉及類胡蘿蔔素合成途徑的關鍵 e-環化酶基因 (LCYe)，CrLCYe 下調株在 -胡蘿蔔素的生產有顯著變化，其產量 2244.3 ug/g，相比於野生型提升了 41.3%。綜合上述，基於 CRISPRi 系統的基因抑制可適用於衣藻 (C. reinhardtii)，並且開拓了提高微藻生產高值化產品的產量，濃度和產率的新技術途徑。

In this study, CRISPRi (clustered regularly interspaced short palindromic repeats interference) was used for the first time to regulate expression of exogenously supplied rfp (red fluorescene protein) gene as a proof-of-concept, and endogenous phosphoenolpyruvate carboxylase (PEPC1) gene as a proof-of-function in Chlamydomonas reinhardtii CC-400. Based on the design of sgRNA binding site, the repression efficiency is about 19%. The application rate of 94% and stability of 7 generations via CRISPRi mediated gene regulation in C. reinhardtii have been demonstrated by RFP. Gene PEPC1 encoding proteins are essential for controlling the carbon flux that enters the TCA cycle and plays a crucial role in carbon partitioning of substrates in competition thus to up-regulate of diglyceride acyltransferase (DGAT) with more lipids synthesis. All CrPEPC1 down-regulated strains have lower chlorophyll colour, but higher biomass concentration and lipid accumulation rate. The outstanding strain generated the highest lipid content and productivity of 28.5% on dry cell weight (DCW) and 34.9 mg/L/day, respectively. This is about 74.4% and 94.2% higher than that of the wild-type. Another gene e-cyclase (LCYe) involving in carotenoid biosynthetic pathway was also tested in the study. The CrLCYe disturbed strains showed significant changes in -carotene production upto 2244.3 ug/g, which yield is 41.3% higher than that of the wide-type. The present results revealed that CRISPRi based transcriptional silencing was applicable in C. reinhardtii and expanded the way to improve the yield, titer and productivity of microalgae-based high-value products.

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