Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR Associated Protein 9（CRISPR/Cas9）系統(tǒng)是近年新興的基因編輯工具之一，另外兩種是ZFN和TALEN。由于CRISPR/Cas9的構(gòu)建更簡易，超越了更早開發(fā)的ZFN和TALEN技術(shù)，成為現(xiàn)今基因編輯領(lǐng)域最炙手可熱的技術(shù)。CRISPR/Cas9技術(shù)源于原核免疫系統(tǒng)CRISPR/Cas。在自然界中，細(xì)菌利用CRISPR/Cas抵抗質(zhì)粒和噬菌體等外源遺傳物質(zhì)的入侵，從而保持自身基因組的完整性。

兩個生物大分子，Cas9蛋白和gRNA（guide RNA）組成了CRISPR/Cas9基因編輯系統(tǒng)。在細(xì)胞內(nèi)，Cas9蛋白與gRNA形成復(fù)合物，能特異性鑒別出靶序列。此過程中，Cas9蛋白負(fù)責(zé)將復(fù)合物定點(diǎn)到靶DNA和剪切靶DNA。Cas9蛋白有6個結(jié)構(gòu)域，分別是Rec I、Rec II、Bridge Helix、PAM Interacting、HNH和RuvC。Rec I是6個中最大的一個結(jié)構(gòu)域，負(fù)責(zé)結(jié)合gRNA。一旦結(jié)合了靶DNA，Bridge Helix就負(fù)責(zé)啟動剪切。PAM interacting結(jié)構(gòu)域賦予Cas9對PAM序列的特異性要求，負(fù)責(zé)啟動與靶DNA的結(jié)合。HNH和RuvC都是核酸酶結(jié)構(gòu)域，剪切單鏈DNA。

圖1. Cas9蛋白結(jié)構(gòu)域 

在沒有結(jié)合gRNA的情況下，Cas9蛋白保持失活狀態(tài)。gRNA是一條單鏈RNA，會形成一個T型結(jié)構(gòu)。在這個T結(jié)構(gòu)里，有幾個顯著的特征：tetraloop、3個stem loop和5’ 端與靶DNA互補(bǔ)的序列。

圖2. gRNA結(jié)構(gòu)

一旦gRNA結(jié)合上Cas9蛋白，就會引起Cas9蛋白的構(gòu)象改變。這種構(gòu)象的改變使得Cas9蛋白從失活狀態(tài)變成了活性狀態(tài)。

圖3. Cas9構(gòu)象改變

一旦Cas9蛋白被激活，它就開始尋找靶DNA。此過程中，它首先結(jié)合到含有PAM（protospacer adjacent motif）序列的序列。一段PAM序列就是2個或者3個堿基序列，位于與gRNA互補(bǔ)的靶DNA序列的下游，緊跟著靶DNA序列。不同的CRISPR系統(tǒng)含有不同的PAM序列，例如：廣泛應(yīng)用的Streptococcus pyogenes Cas9的PAM序列是5′-NGG-3′。當(dāng)Cas9結(jié)合到含有PAM序列的潛在靶DNA序列，它會解開PAM的上游雙鏈DNA序列。此時，gRNA的5’ 端序列與解開的序列配對。一旦配對成功，RuvC和HNH核酸酶結(jié)構(gòu)域就會在PAM序列上游的第3個堿基和第4個堿基之間切開。RuvC剪切下鏈，HNH剪切上鏈。

圖4. Cas9結(jié)合并剪切靶DNA

CRISPRi技術(shù)依靠生成一個沒有核酸酶活性的Cas9蛋白。這是通過往RuvC和NHN兩個核酸酶結(jié)構(gòu)域分別導(dǎo)入氨基酸突變D10A和H840A，使得Cas9蛋白失去切割DNA活性，但仍保留結(jié)合DNA的能力，這樣的Cas9 稱之為dCas9（Dead Cas9）。

圖5. dCas9結(jié)構(gòu)域

當(dāng)dCas9被引導(dǎo)到某個基因的轉(zhuǎn)錄起始位點(diǎn)TSS（transcription start site）時，dCas9能夠物理性阻礙RNA聚合酶的通過，導(dǎo)致基因沉默。為了進(jìn)一步提高轉(zhuǎn)錄抑制的效率，dCas9融合了一個基因抑制結(jié)構(gòu)域，如KRAB（krüppel-associated box）結(jié)構(gòu)域，這樣的蛋白稱之為dCas9-KRAB。此外，dCas9也可以融合雙抑制結(jié)構(gòu)域（bipartite repressor domain）KRAB-MeCP2，抑制效果更佳。

圖6. dCas9-KRAB CRISPRi工作原理

一個完整的dCas9-KRAB CRISPRi慢病毒載體系統(tǒng)包含兩個部分，gRNA表達(dá)載體和dCas9-KRAB（或者dCas9-KRAB-MeCP2）表達(dá)載體。設(shè)計基因抑制實(shí)驗(yàn)時，只需要設(shè)計打靶目的基因的gRNA表達(dá)載體即可。

圖7. dCas9-KRAB CRISPRi慢病毒載體系統(tǒng)

關(guān)于該載體系統(tǒng)的更多信息，請參考以下文獻(xiàn)：

不改變內(nèi)源基因組背景：與CRISPR基因編輯、傳統(tǒng)基因敲除技術(shù)不同，dCas9-KRAB CRISPRi系統(tǒng)不會改變靶基因位點(diǎn)基因組序列。

強(qiáng)基因抑制效果：使用dCas9-KRAB CRISPRi系統(tǒng)進(jìn)行轉(zhuǎn)錄抑制通?？梢垣@得高水平的基因抑制效果。

更多適用的基因種類：由于dCas9-KRAB CRISPRi系統(tǒng)是在DNA水平抑制基因表達(dá)，因此適用于多種轉(zhuǎn)錄本，包括mRNA、非編碼RNA、microRNA、反義轉(zhuǎn)錄本、核定位RNA以及聚合酶III 轉(zhuǎn)錄本的轉(zhuǎn)錄抑制。

特異性：dCas9-KRAB CRISPRi系統(tǒng)可實(shí)現(xiàn)高效抑制同時，幾乎沒有脫靶現(xiàn)象。

不同基因之間差異性：由于dCas9-KRAB需要接觸到目的基因的調(diào)控序列，因此會因?yàn)榛蛩幦旧w位置不同而產(chǎn)生不同的抑制程度，這取決于它們的內(nèi)源染色質(zhì)狀態(tài)。

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of 5'LTR-ΔU3 LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear import of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

U6 promoter: This drives high level expression of the downstream user-selected gRNA.

gRNA: Allows in vitro transcription for RNA preparation. Scaffold gRNA sequence is included.

Terminator: Terminates transcription of the gRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's gene of interest on the vector, leading to their higher expression levels.

3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (since the 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.