近年來，微藻的開發與應用備受矚目，可用於生產多種高價值產品，因此研究極具前景。這十年來，由於基因編輯技術的蓬勃發展，許多研究致力於使用這些新興的技術在微藻基因工程上，以提高其油脂、色素、蛋白質、活性物質等之產量。相較於ZFN及TALEN，CRISPR/Cas9系統擁有高編輯效率、易於操作、低成本，且可以同時進行多組基因編輯的功能，因而被廣泛運用在微藻之相關研究。
本研究第一部分為找出最適合小球藻的電穿轉植載體，嘗試幾次使用衣藻適用之質體於小球藻的電穿轉植失敗後，我們證明使用來自農桿菌帶有左右同源臂 (Left and right border) 及綠色螢光蛋白的質體，在電壓360 V、電容25 F及電阻200 ohm下，能夠增加小球藻的電穿轉化效率。透過分光光度計與倒立螢光顯微鏡之觀測，在Chlorella vulgaris中有兩株綠色螢光表達量高於原藻（28%與67%）；而在Chlorella sorokiniana中則有三株（46%、58%與62%）。
接下來我們將CRISPR/Cas9系統分別應用到Chlorella vulgaris, Chlorella sorokiniana與Chlorella variabilis NC64A。在C. vulgaris中，以帶有左右同源臂的載體pHSE401輔以CRISPR技術干擾fad3基因，使油脂產量較原藻提升高達40%。另外在C. variabilis的研究中，其全基因定序信息完整，能成功獲得PEPC基因，唯其電穿後修復能力不佳，導致後續研究無法進行。目前多數微藻由於基因組訊息尚不完整但藻體本身富涵高GC含量，故設計具有大量鳥糞嘌呤鹼基的sgRNA，命名為輔助調適基因調控 (ASGARD)，分別搭配CRISPRi與CRISPRa系統送入C. sorokiniana中，並針對其生物量、油脂以及蛋白產量分析觀察。雖然轉植藻株之生物量與油脂產量與野生型相比並無明顯差異性，其蛋白產量卻有顯著增加，並可提升超過60%，說明目前開發技術具有顯著的潛力。

Being the green gold of the future, microalgae have recently attracted considerable interest worldwide, for they can be used to produce different kinds of metabolites, such as lipids, protein, pigments and bioactive compounds. In the last decade, the efforts attended to enhance high-value compounds in microalgae are motivated by genetic manipulation. Among them, the CRISPR/Cas9 system appears to be the most efficient and novel technology. Compared to ZFN and TALEN, this newly developed gene editing tool was easier to work with, more affordable and was able to regulate multiple genes simultaneously. In this study, we aimed to enhance the lipid accumulation in three different species of Chlorella by CRISPR/Cas9-based gene editing tools and other characterization analyses, such as growth rate measurement and protein production, had been carried out as well.
In the first part of this study, we attempted to determine plasmid with suitable genomic components which could be utilized in the transformation of Chlorella via electroporation. We had tested several plasmids which could be used in microalgae Chlamydomonas with hsp70A/rbcS and CaMV35S promoter, respectively. However, these plasmids were failed to transform into Chlorella. Finally, plasmid pCAMBIA1302 containing right border (RB), left border (LB) from Agrobacterium tumefaciens and a fragment of mGFP was successfully transformed into microalgae Chlorella vulgaris and Chlorella sorokiniana by electroporation under 360 V at 25 F and 200 ohm. Selected colonies were tested by spectrophotometer and inverted fluorescence microscopy (IFM) with detection at excitation 488 nm and emission 510 nm. Strain 29 and 48 from C. vulgaris as well as strain 2, 4 and 11 from C. sorokiniana showed higher fluorescent value compared with wild type (28% and 67% in C. vulgaris, 46%, 58% and 62% in C. sorokiniana, respectively), proved plasmid with RB/LB is suitable for gene insertion in Chlorella.
Aside from that, pHSE401 containing fragment of Cas9, RB/LB was used as vector, with sgRNA designed from fad3 gene which can affect the lipid accumulation in C. vulgaris. The lipid content of transgenic strains was improved up to 40%. On the other hand, Chlorella variabilis NC64A had been completely sequenced and certain gene like PEPC has been located in its genomic DNA. However, its low recovery rate hampered the transformation efficacy and we were not able to proceed its genome editing. Due to the lack of genomic information in C. sorokiniana and consider most microalgae are in high guanine contents, we establish a new approach via high guanine in sgRNA which name “Adaptive Single Guide Assisted Regulation DNA (ASGARD)”. Coupled with the CRISPRi and CRISPRa system, characterization analyses were conducted to understand how it affect the biomass, lipid accumulation and protein productivity. Although there was no significant difference between wild type and transformants, the protein content of all the transgenic strains was improved up to 60%, g/g DCW. This novel idea and technology are high potential for gene regulating in microalgae.

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