CRISPR

AccuTool ™|Accurate Tool of CRISPR-Cas9 System

Global Leader in Genome Editing Technology

CRISPR-Cas9 technology is a powerful 3rd generation genome editing tool that has improved performance to be more economical and efficient than the 1st and 2nd genome editing tools: Zinc Finger Nuclease (ZFN) and Transcription Activator-Like Effector Nuclease (TALEN).
The CRISPR-Cas9 system uses a guide RNA and a Cas9 nuclease. While the former binds accurately to its specific target DNA region, the latter cuts it, resulting in a site-specific double-strand breaks (DSBs). Genomes can be manipulated while they are being repaired.
AccuTool™ is a genome editing tool developed after the collaboration with ToolGen, being one of the leading companies when it comes to genome editing. BIONEER offers not only Cas9 nuclease, but also design, synthesis, and validation services for guide RNA.

Overview

Derived from adaptive immune systems of a microbiome, CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 genome editing tool cuts the target DNA region and allows the DNA to be repaired naturally. It is also known as "RGENs" (RNA-guided engineered nuclease), as it consists of guide RNA (gRNA) and Cas9 nuclease. This technology provides easier and more efficient ways for precise manipulation of genomes. gRNA consists of crRNA and tracrRNA. The crRNA has a 20-nt target-complementary sequence, while the tracrRNA has the recognition sequence necessary for Cas9-binding (Figure 1). The gRNA guides Cas9 nuclease to the target DNA region, which binds to the complementary sequence in the target DNA region. During this step, the target DNA must contain a PAM (Protospacer adjacent motif) sequence at the 3’-end. After the gRNA forms a complex with the Cas9 nuclease and binds to the target DNA region with the complemtary base pairing, the Cas9 nuclease will recognize the PAM sequence, which results in DSBs at the 3 bp upstream of the PAM sequence (Figure 2).

Figure 1. Diagram of gRNA and Cas9 nuclease complex
The gRNA consists of crRNA and tracrRNA. While the former contains a 20-nt target-complementary sequence, the latter contains a scaffold sequence allowing to form a complex with the Cas9 nuclease.
Figure 2. A schematic diagram of CRISPR-Cas9 System
RNA-guided Cas9 nuclease recognizes the PAM sequence (5'-'NGG'-3') at the 3'-end of the target DNA region and creates DSBs at the 3 bp upstream of the PAM sequence.

DSBs are repaired by means of two pathways: Non-homologous end-joining (NHEJ) or Homology-directed repair (HDR) pathways. If a donor template is not present, DSBs will be repaired through NHEJ pathway. The NHEJ pathway causes a gene knock-out by frameshifts and premature stop codons created from the indels, insertions and deletions of bases in a gene.
On the other hand, when donor templates are present, HDR pathway will take place. Compared with the other case, they can provide more accurate modification and insertion of new genes (Figure 3).

Figure 3. A step-by-step diagram showing CRISPR-Cas9-mediated DSBs repair mechanisms
There are two types of pathways for repairing DSBs: NHEJ and HDR. NHEJ pathway creates an indels, variations by insertions and deletions of few bases during the repair of DSB’s terminal. On the other hand, the HDR pathway uses homologous sequences of donors, either in the form of dsDNA or ssDNA, which allows more accurate gene insertion than the other.

CRISPR-Cas9 Experimental Guide

Knock-out

Knock-in

 STEP 1 
 The first step of genome editing is designing the gRNAs, which guides the Cas9 nuclease to the target DNA region.
STEP 2 

Among the methods to repair the DNA cut by RNA-guided Cas9 nuclease, if you wish to use the NHEJ pathway, the most suitable gRNA and Cas9 nucleases must be decided and synthesized.

    RNP (AccuTool™ aRGEN)
    Plasmid (AccuTool™ dRGEN)
    2-Part gRNA (AccuCRISPR™)

STEP 2 

In order to use HDR, an another pathway of repairing the cut DNA, not only the gRNA and Cas9 nuclease but also the donor template must be designed. After that comes donor synthesis.

In order to use the HDR pathway together with synthesized donor DNA, the types of gRNA and Cas9 nuclease suitable for the experiment are determined and synthesized.

    RNP (AccuTool™ aRGEN)
    Plasmid (AccuTool™ dRGEN)
    2-Part gRNA (AccuCRISPR™)

       
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If you are conducting a knock-out experiment for the first time, we recommend the AccuTool™ CRISPR-Cas9 Starter Kit, an All-In-One Kit that contains all the materials and protocols required for the experiment.

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If you want to insert a desired gene into human AAVS1 gene site or mouse ROSA26 gene site using verified gRNA and HDR donor vector, AccuTool™ Safe Harbor Knock-In Kit is recommended.

       
  STEP 3 
  Precise modification is performed by transfection into target cells with gRNA and Cas9 nuclease.
+ If you want to transfect plasmid forms of Cas9 and sgRNA into cells, we recommend AccuFect™ Transfection Reagent.
  STEP 4 
  Finally, Validation is done to assess the gene editing efficiency using our Mutation Detection kit or In/del analysis service (NGS).

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