CRISPRs/Cas9 技術(shù)是基因組編輯領(lǐng)域重要的突破。在Cas9與sgRNA的共同作用下，定點(diǎn)基因組改變的難度顯著降低，而且可進(jìn)行操作的目標(biāo)生物種類大大增加。Takara旗下Clontech的Guide-It產(chǎn)品線，全面優(yōu)化了目前CRISPRs/Cas9的操作步驟，為成功的基因編輯實(shí)驗(yàn)保駕護(hù)航。

對(duì)于一些難轉(zhuǎn)染的細(xì)胞系，借助慢病毒包裝的方法導(dǎo)入sgRNA和Cas9基因，進(jìn)而實(shí)現(xiàn)靶向基因編輯。但是慢病毒的整合特性，可能會(huì)造成Cas9的恒定表達(dá)。2016年，Takara旗下Clontech新推出的Lenti-X™ Tet-On® 3G CRISPR/Cas9 System，結(jié)合Tet-On 3G 反式轉(zhuǎn)錄激活蛋白質(zhì)，有效規(guī)避了這一擔(dān)憂，通過(guò)添加多西霉素（doxycycline）控制Cas9的表達(dá)，減少可能造成的毒性與脫靶效應(yīng)。

 
Lenti-X CRISPR/Cas9 System是一個(gè)完整的由慢病毒介導(dǎo)的CRISPR/Cas9基因編輯系統(tǒng)。該系統(tǒng)包括Lenti-X Packaging Single Shots（用于產(chǎn)生高滴度的病毒）和三個(gè)不同的質(zhì)粒載體（用于在靶細(xì)胞中表達(dá)Cas9、客戶自定義的sgRNA和Tet-On 3G 反式轉(zhuǎn)錄激活蛋白質(zhì)）。表達(dá)sgRNA的質(zhì)粒系統(tǒng)pLVX-hyg-sgRNA1，易于插入客戶自定義的sgRNA 序列，并且該系統(tǒng)提供足夠的質(zhì)粒供客戶構(gòu)建10種不同的sgRNA序列質(zhì)粒。Tet-On 3G 反式轉(zhuǎn)錄激活蛋白質(zhì)，這使得用戶可以通過(guò)添加多西霉素（doxycycline）來(lái)誘導(dǎo)Cas9的表達(dá)。通過(guò)嚴(yán)格控制Cas9的表達(dá)，Lenti-X Tet-On 3G CRISPR/Cas9 System可以使用戶在細(xì)胞培養(yǎng)中，減少與Cas9恒定表達(dá)相關(guān)的毒性與脫靶效應(yīng)。

 
Vector components for Lenti-X Tet-On 3G CRISPR/Cas9 System.

pLVX-EF1a-Tet3G encodes the Tet-On 3G transactivator protein under the control of the constitutive EF-1 alpha promoter. In contrast with the commonly used CMV promoter, EF-1 alpha is less susceptible to silencing by certain cell types (including various types of stem cells), enabling the establishment of inducible cell lines that are suitable for long-term use. Tet-On 3G is a modified form of the Tet-On Advanced transactivator protein, which was evolved to have a far higher sensitivity to doxycycline. pLVX-TRE3G-Cas9-puro encodes a modified version of the Cas9 endonuclease under the control of the inducible PTRE3GV promoter. The Cas9 sequence in pLVX-TRE3G-Cas9-puro was originally derived from the bacterium S. pyogenes, and has been codon optimized for expression in mammalian cells. Additional Cas9 modifications include the incorporation of a C-terminal nuclear localization signal (NLS). pLVX-hyg-sgRNA1 encodes an sgRNA (designed and inserted by the user) under the constitutive human U6 promoter.

（文中涉及圖片均來(lái)源于Takara Bio USA, Inc.）

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