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與靶位點通過互補配對使Cas9定位到靶序列上，然后切割基因組中的靶位點。

dCas9-KRAB系統(tǒng)是一種用于內(nèi)源性基因轉(zhuǎn)錄抑制的強大工具。該技術(shù)依靠生成一個沒有核酸酶活性的Cas9蛋白。向Cas9的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）。

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我們的慢病毒dCas9-KRAB載體使用第三代慢病毒載體系統(tǒng)。經(jīng)優(yōu)化，該載體在大腸桿菌體內(nèi)具有很高的拷貝數(shù)，包裝的活病毒具有很高的滴度，對大多數(shù)宿主細(xì)胞具有高效的轉(zhuǎn)導(dǎo)能力，能有效地把載體整合到靶細(xì)胞基因組并實現(xiàn)外源基因的高水平表達。人U6啟動子可以高水平驅(qū)動針對靶基因組DNA位點的用戶設(shè)計的gRNA組成型表達。

圖1 基于慢病毒的CRISPRi基因抑制表達效果。（A）轉(zhuǎn)錄阻遏復(fù)合物示意圖。首先向Jurkat細(xì)胞轉(zhuǎn)導(dǎo)攜帶dCas9:KRAB:MeCP2的慢病毒以表達轉(zhuǎn)錄阻遏復(fù)合物，然后進行抗性篩選。然后用另一種慢病毒轉(zhuǎn)導(dǎo)細(xì)胞以表達scramble或者gRNA，并進行額外的抗性篩選。（B） 靶向CXCR4基因啟動子區(qū)的gRNA。（C） 通過qRT-PCR測量CXCR4的相對基因表達，量化了使用Scramble、gRNA以及空白對照（NC）轉(zhuǎn)導(dǎo)的并經(jīng)過抗性篩選的Jurkat細(xì)胞中CXCR4轉(zhuǎn)錄產(chǎn)物表達量。Mean±SD，***P<0.001，****P<0.0001，Tukey事后檢驗。（D） 通過western blot證實了含有打靶g(shù)RNA的Jurkat細(xì)胞中的CXCR4表達水平下調(diào)。

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基因組序列。

強基因抑制效果：使用dCas9-KRAB CRISPRi系統(tǒng)進行轉(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，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)可實現(xiàn)高效抑制同時，幾乎沒有脫靶現(xiàn)象。

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。

需要使用多個載體：使用該載體系統(tǒng)需要與gRNA共表達dCas9-KRAB或者dCas9-KRAB-MeCP2，并且通常以分開的載體表達這些組份。

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，因此會因為基因所處染色體位置不同而產(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)。

Single-gRNA expression lentiviral vector

CMV promoter: Human cytomegalovirus immediate early 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 CMV 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: Drives expression of the user-selected downstream gRNA sequence. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

gRNA: Guide RNA compatible with Cas9 derived from Streptococcus pyogenes.

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 (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.