手机验证
云舟生物科技(广州)股份有限公司
提供定制载体
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。我们的一体化腺病毒CRISPR载体通过表达来自单个腺病毒载体的Cas9和所需的gRNA序列,有助于规避上述挑战。
腺病毒CRISPR载体是一种高效的病毒载体,用于将腺病毒介导的Cas9和靶位点特异性gRNA序列引入多种哺乳动物细胞类型中,其中载体保持为游离体DNA,而不整合到宿主基因组中。它是体内首要选的基因传递系统,常用于基因治疗和疫苗接种。
腺病毒CRISPR载体首先在大肠杆菌中构建为质粒。在载体构建过程中,在两个倒置末端重复序列(ITR)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。然后将载体转染到包装细胞中,其中ITR之间的载体区域被包装成活病毒。当病毒被添加到靶细胞中时,DNA货物被传递到细胞中,在那里它进入细胞核并保持为游离体DNA,而不整合到宿主基因组中。在载体构建过程中放置在两个ITR之间的gRNA和Cas9表达盒与病毒基因组的其余部分一起被引入靶细胞中。
我们的腺病毒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 ITR 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,驱动对两个感兴趣的基因组靶位点特异性 gRNA 序列的表达。
根据设计,腺病毒载体缺乏E1A,E1B和E3基因(delta E1 + delta E3)。前两个是生产活病毒所必需的(这两个基因被设计到包装细胞的基因组中)。因此,由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)。
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
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.
CBh promoter: Chicken beta-actin promoter. Drives expression of the downstream Cas9 nuclease.
Cas9 protein: The open reading frame of the Cas9 nuclease is placed here.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
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 #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.
CBh promoter: Chicken beta-actin promoter. Drives expression of the downstream Cas9 nuclease.
Cas9 protein: The open reading frame of the Cas9 nuclease is placed here.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.
风险提示:丁香通仅作为第三方平台,为商家信息发布提供平台空间。用户咨询产品时请注意保护个人信息及财产安全,合理判断,谨慎选购商品,商家和用户对交易行为负责。对于医疗器械类产品,请先查证核实企业经营资质和医疗器械产品注册证情况。
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 |
科学代表 9:277 (2019) | 使用一体化腺病毒载体靶向 CRISPR/Cas9 |