SgRNA Design and Confirmation

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SgRNA Design and Confirmation    

CRISPR/Cas9 PlatformCB provides a series of sgRNA service, including sequence design, synthesis, validation of efficiency and off-target analysis. In the past decade, our talented scientists are focusing on the development of gene-editing technology and are following the latest technique in this field. Our staff is experienced in sgRNA design and efficiency analysis. We will work closely with you to bring you satisfied services and products.

Since the first description of CRISPR system, it becomes the most acceptable gene-editing tool in the world. The key components of CRISPR system are Cas9 nucleases and guide RNA. In term of targeting different sites genome, sgRNA varies the sequence to each target. A high-quality sgRNA is the foundation of successful gene-editing process. When to select the target sites of Cas9, the only requirement is the presence of PAM. There are multiple type Cas9 nucleases which require different PAMs. The most common used Cas9 nucleases are spCas9 and saCas9 and the PAM are ‘NGG’ and ‘NNGRRT’ respectively. By introducing the target sequence into sgRNA, the Cas9/sgRNA complex can specifically recognize the target sites. Cas9 introduces DNA double strand break after recruited to the specific site. Subsequently, DNA repair may induce random mutant resulting in gene inactivation.

Fig 1. Design sgRNA for spCas9 and saCas9 nucleases1

CRISPR/Cas9 PlatformCB brings customers a set of sgRNA service with high quality, including sgRNA design, construction and validation. Additionally, off-target analysis is available. We provide one of these services separately or a combined service depending on your needs. Before ordering, customers could contact us with your project for free consultation. We will offer feasible analysis for you on a ‘case by case’ basis. Once a project begins, our staff will work closely with you at all the time and keep in touch for each step. We are working to assist you for your goals.

ItemDescriptionTime
sgRNA design

Design sgRNA based on sequence analysis (at least 3 sgRNA)

~1 week

sgRNA construction
  1. SgRNA synthesis
  2. Vector construction (optional)
2-4 week
sgRNA validation
  1. Cell-based Assay
    Transfect sgRNA into HEK293T cells together with Cas9, and then collect cells for sequencing analysis.
  2. Cell-free Assay
    Cas9 protein and sgRNA are mixed with genome DNA in a tube. Then incubate for a certain time and perform sequence analysis to this sample.
2-4 week
Off-target analysis (optional)Varies assays are available for this analysis in terms of different needs, including PCR-based assay, GUIDE-seq, BLESS and Digenome-seq.4-8 week

For more information, don not hesitate to contact us at or 1-631-626-9181. If you could not find what you really want in the website, please feel free to contact us.

Reference

  1. Maximilian, H., et al. (2016) ‘Genome Editing with CRISPR-Cas9: Can It Get Any Better?’, Journal of Genetics and Genomics, 453

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