CRISPR-Cas系統(tǒng)是繼鋅指核酸酶(ZFNs)和TALEN核酸酶之后的另一個(gè)可精確定點(diǎn)編輯基因組DNA的新技術(shù)，具有設(shè)計(jì)構(gòu)建簡單快速等優(yōu)點(diǎn)。已在人類細(xì)胞系、斑馬魚、小鼠、果蠅和酵母等多個(gè)物種中利用。最近，國內(nèi)外研究人員紛紛利用CRISPR-Cas系統(tǒng)定點(diǎn)突變了水稻、小麥、擬南芥等多種作物的特定基因，都證實(shí)CRISPR-Cas系統(tǒng)能夠用于植物的基因組編輯。與ZFN 和TALEN 的設(shè)計(jì)復(fù)雜和各種實(shí)驗(yàn)室試驗(yàn)相比，crispr或許相對(duì)簡單點(diǎn)。crispr／Cas系統(tǒng)允許個(gè)性化的小的非編碼RNA來介導(dǎo)靶標(biāo)基因組DNA的切割，從而使基因能夠通過非同源末端結(jié)合和同源接到的修復(fù)機(jī)制來修飾基因。圖1是利用CRISPR系統(tǒng)敲除植物特定基因的流程簡圖。

圖1

    CRISPR/Cas由20個(gè)堿基的guide RNA 通過Waston-Crick配對(duì)來識(shí)別DNA靶位點(diǎn)序列，從而介導(dǎo)基因的編輯。但是20個(gè)堿基guide RNA也可能與高度同源的DNA序列結(jié)合，從而造成脫靶的問題，這種潛在問題會(huì)限制該技術(shù)在實(shí)際農(nóng)業(yè)生產(chǎn)上的應(yīng)用。但中科院等研究團(tuán)隊(duì)在轉(zhuǎn)基因植株中細(xì)致的分析了靶位點(diǎn)的高度同源位點(diǎn)，并未發(fā)現(xiàn)他們跟靶位點(diǎn)一樣發(fā)生突變。同時(shí)，經(jīng)過高通量測序，在整個(gè)基因組水平上也未發(fā)現(xiàn)有脫靶問題。據(jù)此，他們認(rèn)為在植物系統(tǒng)中CRISPR/Cas并不像鳥槍或者集束炸彈那樣會(huì)有脫靶問題，而是像巡航導(dǎo)彈一般準(zhǔn)確命中目標(biāo)，同時(shí)避免了傷害無辜。

    植物CRISPR專家系統(tǒng)服務(wù)于廣大農(nóng)業(yè)科研人員，本系統(tǒng)基于農(nóng)桿菌轉(zhuǎn)化系統(tǒng)，含一個(gè)表達(dá)Cas9的二元穿梭載體和一個(gè)表達(dá)sgRNA的中間載體，二元穿梭載體質(zhì)粒架構(gòu)基于植物表達(dá)載體pCambia 1303，sgRNA表達(dá)質(zhì)粒啟動(dòng)子為水稻U3 promoter，Cas9基因?yàn)樗久艽a子優(yōu)化且?guī)蓚€(gè)NLS。

    根癌農(nóng)桿菌是一種能誘發(fā)植物產(chǎn)生腫瘤的細(xì)菌，根癌農(nóng)桿菌中誘導(dǎo)植物產(chǎn)生腫瘤的質(zhì)粒（Tumor inducing plasmid），簡稱為 Ti 質(zhì)粒。野生型農(nóng)桿菌的 Ti 質(zhì)粒，含有兩個(gè)與致瘤有關(guān)的區(qū)域：一個(gè)是 T－DNA 區(qū)（transferred DNA region），含致瘤基因；另一個(gè)是毒性區(qū)（Virulence region），在 T－DNA 的切割、轉(zhuǎn)移與整合過程中起作用。用于植物基因轉(zhuǎn)化的農(nóng)桿菌 Ti 質(zhì)粒載體系統(tǒng)的構(gòu)建，是將野生 Ti質(zhì)粒中的致瘤基因刪除，并在 T－DNA 區(qū)域內(nèi)插入適當(dāng)?shù)倪x擇標(biāo)記和多克隆位點(diǎn)。圖2、3是sgRNA表達(dá)質(zhì)粒載體圖；圖4是植物表達(dá)載體 pPL-rCas9的 DNA 圖譜，以 CaMV35S 啟動(dòng)子驅(qū)動(dòng)的水稻密碼子優(yōu)化的Cas9基因以潮霉素抗性為選擇標(biāo)記基因，以β－葡萄糖苷酶（β－glucuronidase,Gus）為報(bào)告基因，經(jīng)此質(zhì)粒轉(zhuǎn)化的獲得的轉(zhuǎn)基因細(xì)胞、組織或植株，具有抗卡那霉素的特性，經(jīng)組織化學(xué)染色呈藍(lán)色。當(dāng)構(gòu)建好sgRNA質(zhì)粒后只要用Kpn I單酶切后一步連接入pPL-rCas9質(zhì)粒即可用于下游的植物CRISPR基因敲除工程操作。

1、構(gòu)建sgRNA表達(dá)載體

圖2

2、Kpn I單酶切線性化pPL-sgRNA載體

圖3

3、插入到經(jīng)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(zhì)粒。

圖4

4、轉(zhuǎn)化農(nóng)桿菌及其下游基因工程篩選操作

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CRISPR/Cas9相關(guān)產(chǎn)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(zhuǎn)錄產(chǎn)物

• sgRNA序列設(shè)計(jì)

• sgRNA表達(dá)載體構(gòu)建

• sgRNA體外合成

• 全基因組sgRNA文庫的構(gòu)建

• sgRNA活性檢測

• Surveyor法及測序驗(yàn)證

• 穩(wěn)定表達(dá)Cas9蛋白細(xì)胞株篩選

• 利用CRISPR系統(tǒng)進(jìn)行基因重組donor質(zhì)粒構(gòu)建

• 植物CRISPR

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