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dCas9-KRAB CRISPRi(CRISPR interference)慢病毒载体系统

概述

CRISPR/Cas9载体属于几种新兴的基因组编辑工具之一,可以快速有效地在基因组的靶位点产生突变(另外两种应用广泛的是ZFN和TALEN)。

Cas9属于RNA引导的DNA核酸酶,是天然原核免疫系统的一部分,赋予细菌对质粒和噬菌体等外源遗传物质的抗性。在细胞内,Cas9核酸酶与引导RNA(gRNA)形成复合物,该复合物通过与基因组中的18-22 nt的同源靶序列直接相互作用提供靶向特异性。gRNA与靶位点通过互补配对使Cas9定位到靶序列上,然后切割基因组中的靶位点。

dCas9-KRAB系统是一种用于内源性基因转录抑制的强大工具。该技术依靠生成一个没有核酸酶活性的Cas9蛋白。向Cas9的RuvC和NHN两个核酸酶结构域分别导入氨基酸突变D10A和H840A,使得Cas9蛋白失去切割DNA活性,但仍保留结合DNA的能力,这样的Cas9 称之为dCas9(Dead Cas9)。

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当dCas9被引导到某个基因的转录起始位点TSS(transcription start site)时,dCas9能够物理性阻碍RNA聚合酶的通过,导致基因沉默。为了进一步提高转录抑制的效率,dCas9融合了一个基因抑制结构域,如KRAB(krüppel-associated box)结构域,这样的蛋白称之为dCas9-KRAB。此外,dCas9也可以融合双抑制结构域(bipartite repressor domain)KRAB-MeCP2,抑制效果更佳。

一个完整的dCas9-KRAB CRISPRi慢病毒载体系统包含两个部分,gRNA表达载体和dCas9-KRAB(或者dCas9-KRAB-MeCP2)表达载体。设计基因抑制实验时,只需要设计打靶目的基因的gRNA表达载体即可。

关于该载体系统的更多信息,请参考以下文献:

References Topic
Cell. 154:442 (2013) Characterization of CRISPRa and CRISPRi systems
Nat Methods. 12:1143 (2015) Characterization of the dCas9-KRAB system
Nat Methods. 15:611 (2018) Dual-gRNA and dCas9-KRAB-MeCP2 based gene repression
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亮点

我们的慢病毒dCas9-KRAB载体使用第三代慢病毒载体系统。经优化,该载体在大肠杆菌体内具有很高的拷贝数,包装的活病毒具有很高的滴度,对大多数宿主细胞具有高效的转导能力,能有效地把载体整合到靶细胞基因组并实现外源基因的高水平表达。人U6启动子可以高水平驱动针对靶基因组DNA位点的用户设计的gRNA组成型表达。

试验验证

图1 基于慢病毒的CRISPRi基因抑制表达效果。(A)转录阻遏复合物示意图。首先向Jurkat细胞转导携带dCas9:KRAB:MeCP2的慢病毒以表达转录阻遏复合物,然后进行抗性筛选。然后用另一种慢病毒转导细胞以表达scramble或者gRNA,并进行额外的抗性筛选。(B) 靶向CXCR4基因启动子区的gRNA。(C) 通过qRT-PCR测量CXCR4的相对基因表达,量化了使用Scramble、gRNA以及空白对照(NC)转导的病经过抗性筛选的Jurkat细胞中CXCR4转录产物表达量。Mean±SD,***P<0.001,****P<0.0001,Tukey事后检验。(D) 通过western blot证实了含有打靶gRNA的Jurkat细胞中的CXCR4表达水平下调。

优势

不改变内源基因组背景:与CRISPR基因编辑、传统基因敲除技术不同,dCas9-KRAB CRISPRi系统不会改变靶基因位点基因组序列。

强基因抑制效果:使用dCas9-KRAB CRISPRi系统进行转录抑制通常可以获得高水平的基因抑制效果。

更多适用的基因种类:由于dCas9-KRAB CRISPRi系统是在DNA水平抑制基因表达,因此适用于多种转录本,包括mRNA、非编码RNA、microRNA、反义转录本、核定位RNA以及聚合酶III 转录本的转录抑制。

特异性:dCas9-KRAB CRISPRi系统可实现高效抑制同时,几乎没有脱靶现象。

不足之处

技术复杂:使用慢病毒载体时,需要在包装细胞中产生活病毒,然后测定病毒滴度。因此慢病毒转染相对于常规质粒转染,技术难度更高,周期更长。

需要使用多个载体:使用该载体系统需要与gRNA共表达dCas9-KRAB或者dCas9-KRAB-MeCP2,并且通常以分开的载体表达这些组份。

不同基因之间差异性:由于dCas9-KRAB需要接触到目的基因的调控序列,因此会因为基因所处染色体位置不同而产生不同的抑制程度,这取决于它们的内源染色质状态。

载体关键元件
Single-gRNA expression lentiviral vector

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: 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.

Dual-gRNA gRNA expression lentiviral vector

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, 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 RSV 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 importation 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 #1: The first guide RNA compatible with Cas9 derived from Streptococcus pyogenes.

gRNA #2: The second guide RNA compatible with Cas9 derived from Streptococcus pyogenes.

Terminator: Terminates transcription of the gRNA.

hPGK promoter: Human phosphoglycerate kinase 1 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 viral RNA stability in packaging cells, leading to higher titer of packaged virus.

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 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.