CRISPRs/Cas9 技術是基因組編輯領域重要的突破。在Cas9與sgRNA的共同作用下，定點基因組改變的難度顯著降低，而且可進行操作的目標生物種類大大增加。Takara旗下Clontech的Guide-It產品線，全面優化了目前CRISPRs/Cas9的操作步驟，為成功的基因編輯實驗保駕護航。

對于一些難轉染的細胞系，借助慢病毒包裝的方法導入sgRNA和Cas9基因，進而實現靶向基因編輯。但是慢病毒的整合特性，可能會造成Cas9的恒定表達。2016年，Takara旗下Clontech新推出的Lenti-X™ Tet-On® 3G CRISPR/Cas9 System，結合Tet-On 3G 反式轉錄激活蛋白質，有效規避了這一擔憂，通過添加多西霉素（doxycycline）控制Cas9的表達，減少可能造成的毒性與脫靶效應。

 
Lenti-X CRISPR/Cas9 System是一個完整的由慢病毒介導的CRISPR/Cas9基因編輯系統。該系統包括Lenti-X Packaging Single Shots（用于產生高滴度的病毒）和三個不同的質粒載體（用于在靶細胞中表達Cas9、客戶自定義的sgRNA和Tet-On 3G 反式轉錄激活蛋白質）。表達sgRNA的質粒系統pLVX-hyg-sgRNA1，易于插入客戶自定義的sgRNA 序列，并且該系統提供足夠的質粒供客戶構建10種不同的sgRNA序列質粒。Tet-On 3G 反式轉錄激活蛋白質，這使得用戶可以通過添加多西霉素（doxycycline）來誘導Cas9的表達。通過嚴格控制Cas9的表達，Lenti-X Tet-On 3G CRISPR/Cas9 System可以使用戶在細胞培養中，減少與Cas9恒定表達相關的毒性與脫靶效應。

 
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.

（文中涉及圖片均來源于Takara Bio USA, Inc.）

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