CRISPR-Cas系統是繼鋅指核酸酶(ZFNs)和TALEN核酸酶之后的另一個可精確定點編輯基因組DNA的新技術，具有設計構建簡單快速等優點。已在人類細胞系、斑馬魚、小鼠、果蠅和酵母等多個物種中利用。最近，國內外研究人員紛紛利用CRISPR-Cas系統定點突變了水稻、小麥、擬南芥等多種作物的特定基因，都證實CRISPR-Cas系統能夠用于植物的基因組編輯。與ZFN 和TALEN 的設計復雜和各種實驗室試驗相比，crispr或許相對簡單點。crispr／Cas系統允許個性化的小的非編碼RNA來介導靶標基因組DNA的切割，從而使基因能夠通過非同源末端結合和同源接到的修復機制來修飾基因。圖1是利用CRISPR系統敲除植物特定基因的流程簡圖。

圖1

    CRISPR/Cas由20個堿基的guide RNA 通過Waston-Crick配對來識別DNA靶位點序列，從而介導基因的編輯。但是20個堿基guide RNA也可能與高度同源的DNA序列結合，從而造成脫靶的問題，這種潛在問題會限制該技術在實際農業生產上的應用。但中科院等研究團隊在轉基因植株中細致的分析了靶位點的高度同源位點，并未發現他們跟靶位點一樣發生突變。同時，經過高通量測序，在整個基因組水平上也未發現有脫靶問題。據此，他們認為在植物系統中CRISPR/Cas并不像鳥槍或者集束炸彈那樣會有脫靶問題，而是像巡航導彈一般準確命中目標，同時避免了傷害無辜。

    植物CRISPR專家系統服務于廣大農業科研人員，本系統基于農桿菌轉化系統，含一個表達Cas9的二元穿梭載體和一個表達sgRNA的中間載體，二元穿梭載體質粒架構基于植物表達載體pCambia 1303，sgRNA表達質粒啟動子為水稻U3 promoter，Cas9基因為水稻密碼子優化且帶兩個NLS。

    根癌農桿菌是一種能誘發植物產生腫瘤的細菌，根癌農桿菌中誘導植物產生腫瘤的質粒（Tumor inducing plasmid），簡稱為 Ti 質粒。野生型農桿菌的 Ti 質粒，含有兩個與致瘤有關的區域：一個是 T－DNA 區（transferred DNA region），含致瘤基因；另一個是毒性區（Virulence region），在 T－DNA 的切割、轉移與整合過程中起作用。用于植物基因轉化的農桿菌 Ti 質粒載體系統的構建，是將野生 Ti質粒中的致瘤基因刪除，并在 T－DNA 區域內插入適當的選擇標記和多克隆位點。圖2、3是sgRNA表達質粒載體圖；圖4是植物表達載體 pPL-rCas9的 DNA 圖譜，以 CaMV35S 啟動子驅動的水稻密碼子優化的Cas9基因以潮霉素抗性為選擇標記基因，以β－葡萄糖苷酶（β－glucuronidase,Gus）為報告基因，經此質粒轉化的獲得的轉基因細胞、組織或植株，具有抗卡那霉素的特性，經組織化學染色呈藍色。當構建好sgRNA質粒后只要用Kpn I單酶切后一步連接入pPL-rCas9質粒即可用于下游的植物CRISPR基因敲除工程操作。

1、構建sgRNA表達載體

圖2

2、Kpn I單酶切線性化pPL-sgRNA載體

圖3

3、插入到經Kpn I 單酶切線性化的pPL-rCas9的載體，得到靶向特定基因的CRISPR基因敲除質粒。

圖4

4、轉化農桿菌及其下游基因工程篩選操作

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CRISPR/Cas9相關產品：

• Precut pSG-target Cloning kit & SSA assay

• Different type cas9 expression plasmid CRISPR /Cas9載體

• T7 sgRNA MICscriptTM KIT/sgRNA synthesis product

• Precut SgRNA Cloning kit & pSD-gRNA Plasmid

• EzOmicsTM RNA QUICK clear KIT-------一步純化RNA轉錄產物

CRISPR相關服務：

• sgRNA序列設計

• sgRNA表達載體構建

• sgRNA體外合成

• 全基因組sgRNA文庫的構建

• sgRNA活性檢測

• Surveyor法及測序驗證

• 穩定表達Cas9蛋白細胞株篩選

• 利用CRISPR系統進行基因重組donor質粒構建

• 植物CRISPR

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