CRISPR/Cas9載體屬于幾種新興的基因組編輯工具之一，可以快速有效地在基因組的靶位點產(chǎn)生突變（另外兩種應(yīng)用廣泛的是ZFN和TALEN）。

Cas9屬于RNA引導(dǎo)的DNA核酸酶，是天然原核免疫系統(tǒng)的一部分，賦予細(xì)菌對質(zhì)粒和噬菌體等外源遺傳物質(zhì)的抗性。在細(xì)胞內(nèi)，Cas9核酸酶與引導(dǎo)RNA（gRNA）形成復(fù)合物，該復(fù)合物通過與基因組中的18-22 nt的同源靶序列直接相互作用提供靶向特異性。gRNA與靶位點通過互補(bǔ)配對使Cas9定位到靶序列上，然后切割基因組中的靶位點。

協(xié)同激活介質(zhì)（SAM）系統(tǒng)是用于轉(zhuǎn)錄激活內(nèi)源性基因的強(qiáng)大工具。該系統(tǒng)源自CRISPR/Cas9基因組編輯系統(tǒng)，但不具有基因組編輯功能，是一種可指導(dǎo)在靶位點進(jìn)行多組分轉(zhuǎn)錄激活復(fù)合物（SAM復(fù)合物）組裝的修飾型gRNA。通常，SAM復(fù)合物的組裝足以誘導(dǎo)靶位點強(qiáng)烈的轉(zhuǎn)錄激活。

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該慢病毒gRNA/MS2表達(dá)載體系統(tǒng)利用了gRNA指導(dǎo)的活化復(fù)合物在靶位點組裝的特性，用于內(nèi)源基因組靶位點的轉(zhuǎn)錄激活。

圖1 基于慢病毒的CRISPRa系統(tǒng)的基因上調(diào)表達(dá)效果。（A）SAM復(fù)合物示意圖。首先向NIH3T3細(xì)胞共轉(zhuǎn)導(dǎo)兩種分別攜帶dCas9:VP64和MS2:P65:HSF1的慢病毒，進(jìn)行抗性篩選。然后使用遞送msgRNA的第三種慢病毒轉(zhuǎn)導(dǎo)細(xì)胞，并用另一種抗生素進(jìn)行篩選。（B）針對靶向mBrn2基因啟動子區(qū)域而設(shè)計的msgRNA。（C） 通過qRT-PCR測量mBrn2的相對基因表達(dá)，量化了使用Scramble、msgRNA以及空白對照（NC）轉(zhuǎn)導(dǎo)的并經(jīng)過抗性篩選的NIH3T3細(xì)胞中mBrn2轉(zhuǎn)錄產(chǎn)物表達(dá)量。Mean±SD，*P<0.05，Turkey事后檢驗。

內(nèi)源基因組背景：SAM系統(tǒng)可以激活內(nèi)源基因組靶位點的轉(zhuǎn)錄。與轉(zhuǎn)基因和基因編輯技術(shù)不同，該技術(shù)方法不會涉及改變靶基因位點基因組序列。

獨立的調(diào)控手段: 使用SAM載體系統(tǒng)靶向轉(zhuǎn)錄激活基因并不需要預(yù)先了解目的基因的天然調(diào)控機(jī)理，但需要知道靶位點DNA序列的準(zhǔn)確信息。

強(qiáng)烈激活：使用SAM系統(tǒng)轉(zhuǎn)錄激活的基因通常得到非常高水平的表達(dá)。

技術(shù)復(fù)雜：使用慢病毒載體時，需要在包裝細(xì)胞中產(chǎn)生活病毒，然后測定病毒滴度。因此慢病毒轉(zhuǎn)染相對于常規(guī)質(zhì)粒轉(zhuǎn)染，技術(shù)難度更高，周期更長。

需要多個載體：該載體系統(tǒng)需要gRNA/MS2、MS2/P65/HSF1和dCas9/VP64共表達(dá)，并且這些組分歸屬于不同的載體。

特異性：基于SAM的基因靶向激活方法相對較新，目前還沒有關(guān)于使用gRNA/MS2 RNA靶向特異性的詳細(xì)報道。

CMV promoter: Human cytomegalovirus immediate early promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

Δ5' LTR: 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, Δ5' LTR 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 CMV promoter engineered upstream of Δ5' 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 gRNA.

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

MS2 scaffold: This hairpin aptamer sequence binds robustly to fusion proteins containing the MS2 bacteriophage coat proteins.

Terminator: Terminates transcription of the gRNA.

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

ΔU3/3' LTR: 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 (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR 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.