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CRISPR / Cas9载体是几种新兴的基因组编辑工具之一,可以在基因组的靶位点快速有效地创建突变(另外两种流行的是ZFN和TALEN)。
Cas9是一类RNA引导的DNA核酸酶的成员,它们是天然原核免疫系统的一部分,可赋予对外来遗传元素(如质粒和噬菌体)的抗性。在细胞内,Cas9酶与引导RNA(gRNA)形成复合物,通过与基因组中同源的18-22nt靶序列直接相互作用提供靶向特异性。gRNA与靶位点的杂交定位Cas9,然后切割基因组中的靶位点。
为了实现CRISPR介导的基因靶向,靶细胞必须同时共表达Cas9和靶位点特异性gRNA。这可以通过表达来自同一载体(又名多合一载体)的Cas9和gRNA序列或使用单独的载体来驱动Cas9和gRNA表达(分别为仅Cas9和仅gRNA载体)来实现。使用一体化载体表达Cas9和gRNA的优势在于,它提供了使用单一载体将CRISPR介导的基因编辑所需的所有组分递送到细胞的机会,这在技术上是直接的。使用单独的载体表达Cas9和gRNA需要将靶细胞与两个单独的载体共转导,这在技术上可能具有挑战性,因为并非所有细胞都会同时使用gRNA和Cas9载体转导。使用单独载体的另一种方法是转导具有所需gRNA序列稳定表达高水平Cas9的细胞或生物体。但是,这种方法可能相当耗时且劳动密集。我们的一体化慢病毒CRISPR载体通过表达来自单个慢病毒载体的Cas9和所需的gRNA序列,有助于规避上述挑战。
我们的慢病毒CRISPR载体是一种高效的病毒载体,可将Cas9和靶位点特异性gRNA序列永久引入难以转染的哺乳动物细胞中。慢病毒CRISPR载体首先在大肠杆菌中构建为质粒。在载体构建过程中,在两个长末端重复序列(LTR)之间克隆gRNA和Cas9表达盒。人类U6启动子驱动用户选择的gRNA序列的表达,该序列将Cas9引导至感兴趣的DNA靶位点。然后将载体与几个辅助质粒一起转染到包装细胞中。在包装细胞内,位于LTR之间的载体DNA被转录成RNA,辅助质粒表达的病毒蛋白进一步将RNA包装成病毒。然后将活病毒释放到上清液中,上清液可用于直接感染靶细胞或在浓缩后感染。当病毒被添加到靶细胞中时,RNA货物被穿梭到细胞中,在那里它被逆转录成DNA,并永久整合到宿主基因组中,导致Cas9和用户选择的gRNA序列共表达。
我们的慢病毒CRISPR载体可用于表达单gRNA或双gRNA。虽然单gRNA载体广泛用于传统的CRISPR基因组编辑应用,例如产生单基因敲除,但双gRNA载体对于需要同时靶向一对基因组位点的应用是必要的。此类应用的示例包括:1)配对的Cas9切口酶实验,其中hCas9的“切口酶”突变形式(hCas10-D9A)与靶向单个靶位点的两条相反链的两个gRNA结合使用,以在每条链上产生单链切割一个,从而导致DSB的靶向特异性高于单个gRNA;2)在一对gRNA靶向的两个DSB之间产生片段的缺失;3)同时靶向两个不同的基因。单个 gRNA 载体由单个人 U6 启动子组成,驱动两个 LTR 之间的靶位点特异性 gRNA 序列,而双 gRNA 载体由两个连续的 U6 启动子组成,驱动特定于两个目标基因组靶位点的 gRNA 序列的表达。
根据设计,我们的慢病毒载体缺乏病毒包装和转导所需的基因(这些基因由病毒包装过程中使用的辅助质粒携带)。因此,由慢病毒载体产生的病毒具有复制不全的重要安全特征(这意味着它们可以转导靶细胞但不能在其中复制)。
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: This drives high level expression of the downstream gRNA. 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.
EFS Promoter: Human eukaryotic translation elongation factor 1 α1 short form. Drives expression of the downstream Cas9 nuclease.
Cas9 protein: The open reading frame of the Cas9 nuclease is placed here.
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.
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: This drives high level expression of the 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.
EFS Promoter: Human eukaryotic translation elongation factor 1 α1 short form. Drives expression of the downstream Cas9 nuclease.
Cas9 protein: The open reading frame of the Cas9 nuclease is placed here.
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.
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有关该矢量系统的更多信息,请参阅以下论文。
引用 | 主题 |
---|---|
科学。339:819 (2013) | 使用CRISPR/Cas9系统进行基因组编辑的描述 |
细胞。154:1380–9 (2013) | 使用 Cas9 D10A 双切口提高特异性 |
国家生物技术.31:827 (2013) | RNA引导的Cas9核酸酶的特异性 |
科学。343:84 (2014) | 使用一体化慢病毒载体靶向 CRISPR/Cas9 |