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CRISPRPL

Sigma-Aldrich

CRISPR Plant

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About This Item

UNSPSC Code:
41106609

application(s)

CRISPR

Quality Level

200

shipped in

dry ice

storage temp.

−20°C

Related Categories

General description

All-in-one, ready-to-use Cas9 and guide RNA (gRNA) expression plasmids for use with monocots and dicots.

CRISPR Plant Cas9 products are intended for Agrobacterium-mediated plant transformation or biolistic microparticle bombardment or protoplast transformation. The products are based on the type IIA CRISPR-Cas9 derived from Streptococcus pyogenes. The native Cas9 coding sequence is codon optimized for expression in monocots and dicots, respectively. The monocot Cas9 constructs contain a monocot U6 promoter for sgRNA expression, and the dicot Cas9 constructs contain a dicot U6 promoter.

The plant selection markers include:

  • hygromycin B resistance gene
  • neomycin phosphotransferase gene
  • bar gene (phosphinothricin acetyl transferase)

Application

  • Inactivation of genes
  • Target validation
  • Site specific integration of gene of interest
  • Gene replacement via HR

Features and Benefits

  • Main advantages of CRISPR/Cas9 are in terms of simplicity, accessibility, cost and versatility.
  • CRISPR/Cas9 system does not require any protein engineering steps, making it much more straightforward to test multiple gRNAs for each target gene
  • Only 20 nt in the gRNA sequence need to be changed to confer a different target specificity
  • Another advantage of CRISPR/Cas9 compared to ZFNs and TALENs is the ease of multiplexing. The simultaneous introduction of DSBs at multiple sites can be used to edit several genes at the same time. It can be particularly useful to knock out redundant genes or parallel pathways. Multiplex editing with the CRISPR/Cas9 system simply requires the monomeric Cas9 protein and any number of different sequence-specific gRNAs.
  • CRISPR/Cas9 system can cleave methylated DNA in human cells allowing genomic modifications that are beyond the reach of the other nucleases. This has not been specifically explored in plants, it is reasonable to assume that the ability to cleave methylated DNA is intrinsic to the CRISPR/Cas9 system and not dependent on the target genome

Components

1 vial containing 50ul of 20ng/ul plasmid DNA
Keep reagent tubes closed when not in use.
Practice aseptic lab technique to avoid DNase contamination.

Principle

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be programmed with a single gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB.

Other Notes

For ordering any of our custom CRISPR plant products please visit: CUSTOM ORDERING FORM

Legal Information

CRISPR Use License Agreement

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 1

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

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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. Why did my CRISPR plant construct did not produce the expected target cleavage?

    1 answer

    1. Not every sgRNA will produce the target cleavage. It is recommended to use two to three sgRNAs per gene to increase the successful rate.

      Helpful?

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

    1 answer

    1. Kanamycin.

      Helpful?

  5. 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?

  6. What is the Department of Transportation shipping information for this product?

    1 answer

    1. Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.

      Helpful?

  7. 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?

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

    1 answer

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

      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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