哺乳动物gRNA/MS2表达慢病毒载体

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。

协同激活介质(SAM)系统是用于转录激活内源性基因的强大工具。该系统源自CRISPR/Cas9基因组编辑系统,但不具有基因组编辑功能,是一种可指导在靶位点进行多组分转录激活复合物(SAM复合物)组装的修饰型gRNA。通常,SAM复合物的组装足以诱导靶位点强烈的转录激活。

完整的SAM系统包含三个组分,每个组分分别由单独的慢病毒载体提供:gRNA/MS2表达载体,MS2/P65/HSF1和dCas9/VP64辅助载体。

将用户定制的gRNA序列克隆到gRNA/MS表达载体。在该载体中,经修饰的gRNA包括两个138-nt发夹RNA适体,形成噬菌体MS2衣壳蛋白的结合位点。这些发夹RNA适体与gRNA连接,有利于高效募集MS2-融合蛋白。

MS2/P65/HSF1辅助载体驱动由MS2,p65(NF-kB的反式激活亚基)和HSF1(人热休克因子1的激活结构域)组成的三结构域融合蛋白的表达。

dCas9/VP64辅助载体驱动催化失活变体dCas9和合成型VP64反式激活结构域融合蛋白的表达。

当这三种载体共转导细胞时,用户定制的gRNA可能会募集MS2/P65/HSF1(通过MS2结合发夹适体与gRNA连接)和dCas9/VP64(通过CRISPR/Cas9复合物组装) 到gRNA靶位点,从而组装出强大的SAM复合物。 这些SAM复合物可通过VP64,p65和HSF1激活结构域之间的协同相互作用实现靶位点的强烈转录激活。

该载体系统主要设计用于基因组的大规模筛选,使用gRNA序列文库来产生gRNA/MS2表达载体文库。同时,该系统也可用于激活单个或一系列基因的转录。

关于该载体系统的更多信息,请参考以下文献。

参考文献 主题
Nature. 517:583 (2015) Description of the SAM system
EMBO J. 12:595 (1993) The RNA binding site of bacteriophage MS2 coat protein
Biochem Soc Trans. 36:603 (2008) The p65 activation domain
Redox Biol. 2:535 (2014) The HSF1 activation domain
Proc Natl Acad Sci U S A. 95:14628 (1998) The VP64 activation domain
亮点

该慢病毒gRNA/MS2表达载体系统利用了gRNA指导的活化复合物在靶位点组装的特性,用于内源基因组靶位点的转录激活。

优势

内源基因组背景:SAM系统可以激活内源基因组靶位点的转录。与转基因和基因编辑技术不同,该技术方法不会涉及改变靶基因位点基因组序列。

独立的调控手段: 使用SAM载体系统靶向转录激活基因并不需要预先了解目的基因的天然调控机理,但需要知道靶位点DNA序列的准确信息。

强烈激活:使用SAM系统转录激活的基因通常得到非常高水平的表达。

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。

需要多个载体:该载体系统需要gRNA/MS2、MS2/P65/HSF1和dCas9/VP64共表达,并且这些组分归属于不同的载体。

特异性:基于SAM的基因靶向激活方法相对较新,目前还没有关于使用gRNA/MS2 RNA靶向特异性的详细报道。

载体关键元件

RSV promoter: Rous sarcoma virus 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 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 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.