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CRISPRPL

Sigma-Aldrich

CRISPR 植物

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About This Item

UNSPSC代码:
41106609

应用

CRISPR

运输

dry ice

储存温度

−20°C

相关类别

一般描述

一体化的即用型Cas9和向导RNA（gRNA）表达质粒，可用于单子叶植物和双子叶植物。

CRISPR植物Cas9产品可用于农杆菌属介导的植物转化或基因枪微粒轰击或原生质体转化。该产品是基于酿脓链球菌的IIA型CRISPR-Cas9。天然的Cas9编码序列经过密码子优化，可分别用于在单子叶植物和双子叶植物中进行表达。单子叶植物Cas9构建体含有可驱动sgRNA表达的单子叶植物U6启动子，双子叶植物Cas9构建体含有双子叶植物U6启动子。

植物筛选标记物包括：

  • 潮霉素B抗性基因
  • 新霉素磷酸转移酶基因
  • 抗除草剂基因（草丁膦乙酰转移酶）

应用

  • 基因失活
  • 靶标验证
  • 目的基因的位点特异性整合
  • 通过HR替换基因

特点和优势

  • CRISPR/Cas9的主要优势在于其具有简单性、易用性、低成本和通用性。
  • CRISPR/Cas9系统不需要任何蛋白质工程步骤，更易于检测每个目标基因的多个gRNA
  • 只需改变gRNA序列中的20个核苷酸，即可获得不同的靶点特异性
  • 与ZFN和TALEN相比，CRISPR/Cas9的另一个优势在于它可同时在多个位点导入DSB，从而实现对多个基因同时进行编辑。这对于敲除多余基因或平行通路特别有帮助。使用CRISPR/Cas9系统进行同时编辑，只需单体Cas9蛋白以及任何数量的、不同的序列特异性gRNA。
  • CRISPR/Cas9系统可切割人类细胞中的甲基化DNA，其达到的基因修饰超越了其他核酸酶。这一点尚未在植物中进行探索，可以认为切割甲基化DNA是CRISPR/Cas9系统特有的功能，并且与目标基因组无关。

组分

1管含50μl的20ng/μl质粒DNA
在不使用时，将试管盖扣紧。
利用无菌实验技术，避免DNAase污染。

原理

CRISPR/Cas 系统被细菌和古细菌用作防御入侵病毒和质粒的防御。最近，来自细菌化脓性链球菌的 II 型 CRISPR/Cas 系统已经被设计成使用两种分子组分在真核系统中起作用：单个 Cas9 蛋白和非编码指导 RNA（gRNA）。Cas9 核酸内切酶可以用单个 gRNA 编程，将 DNA 双链断裂（DSB）导向所需的基因组位置。与由锌指核酸酶（ZFN）诱导的 DSB 类似，细胞然后激活内源 DNA 修复过程，非同源末端连接（NHEJ）或同源定向修复（HDR），以愈合目标 DSB。

其他说明

如需订购我们的定制 CRISPR 工厂产品，请访问：定制订购表格

法律信息

CRISPR 使用许可协议

储存分类代码

12 - Non Combustible Liquids

WGK

WGK 1

闪点(°F)

Not applicable

闪点(°C)

Not applicable

历史批次信息供参考:

分析证书(COA)

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Questions

1–10 of 13 Questions  

  1. Can this be used to activate genes rather than inactivation? If so, how can it be used to activate certain genes.

    1 answer

    1. This system may only be used to inactivate genes. This item has an active Cas9 that cuts DNA, causing a deletion or an insertion after the cell repairs the break. This insertion or deletion will result in a frame shift, thereby stopping the correct translation of protein. This is a gene knockout. If a donor DNA template is added, homologous recombination occurs and the donor template is inserted into the DNA. CRISPR activation generally involves a dCas9 (inactive, dead Cas9) that has another functional protein domain fused to it that stimulates RNA transcription. The dCas9 fusion protein for gene activation is generally targeted to the transcription start site (TSS) of genes.

      Helpful?

  2. which strain of Agrobacterium tumefaciens is recommended when using p63, p55, p53 or p54 vector

    1 answer

    1. Please navigate to the link https://www.sigmaaldrich.com/techservice, click on "Product Technical Inquiries" under the Products Section with all the required information so that a member of the Technical Service team can reach out to assist further. Thank you.

      Helpful?

  3. How can I use the CRISPR plant vectors for homologous recombination (HR)?

    1 answer

    1. First, you need to design an HR DNA donor with the target of interest and the homologous arms. For Agrobacterium-mediated plant transformation, the HR DNA donor can be inserted into an appropriate restriction site, such as the EcoRI site or the HindIII site, within the T-DNA borders. For biolistics or protoplast plant transformation, the HR DNA donor can be inserted into XbaI, EcoRI, HindIII, or XhoI site. Alternatively the HR DNA donor can be co-delivered in a separate donor vector.

      Helpful?

  4. What is the promoter used for sgRNA (single guide RNA) expression?

    1 answer

    1. In the monocot CRISPR vectors, it is a wheat U6 promoter. In the dicot CRISPR vectors, it is an Arabidopsis thaliana U6 promoter.

      Helpful?

  5. Can I use the monocot CRISPR plant vectors on dicots plants and the dicot CRISPR plant vectors on monocot plants?

    1 answer

    1. Yes. But it is not recommended. The monocot CRISPR plant vectors are more suited for monocot plants, and the dicots CRISPR plant vectors are more suited for dicot plants.

      Helpful?

  6. What is the promoter used for the expression of the selection markers?

    1 answer

    1. The 2X35S promoter is used in this case.

      Helpful?

  7. What antibiotics should I use for selection of the CRISPR Plant vectors in E. coli or Agrobacteria tumefaciens?

    1 answer

    1. Kanamycin.

      Helpful?

  8. How is the Cas9 codon sequence optimized?

    1 answer

    1. In the monocot CRISPR Plant vectors, the Cas9 codon optimization is based on Zea mays. In the dicot CRISPR Plant vectors, the Cas9 codon optimization is based on Arabidopsis thaliana.

      Helpful?

  9. Can one use an Agrobacterium-mediated transformation CRISPR vector for biolistics or protoplast plant transformation?

    1 answer

    1. Yes. However, the biolistics/protoplast CRISPR vectors cannot be used for Agrobacterium-mediated plant transformation.

      Helpful?

  10. 1. What type of Cas9 is used in the CRISPR Plant vectors?

    1 answer

    1. Type IIA Cas9 derived from Streptococcus pyogenes.

      Helpful?

1–10 of 13 Questions  

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