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Cat. No.: KO13-20

Cat. No.: KO15-20

Cat. No.: KO23-20

Description

CRISPR-Cas9 gene editing technology works by directing the Cas9 enzyme to the target DNA sequence using guide RNA (gRNA). The Cas9 enzyme then cuts the target DNA double strand, causing a DNA double-strand break. This break is repaired incorrectly by non-homologous end joining (NHEJ), leading to gene knockout.

Traditional methods of constructing CRISPR-Cas9 plasmids involve plasmids that can be as large as 6-10 kb, making them difficult to transfect. The transfection efficiency is even lower in hard-to-transfect cells, such as primary cells. Additionally, plasmids continuously produce CRISPR-Cas9, which significantly increases the off-target rate.

The chemical synthesis of sgRNA and the transfection of RNP (ribonucleoprotein) complexes, on the other hand, optimize the CRISPR-Cas9 components. This approach improves transfection efficiency, reduces cell toxicity, lowers off-target effects, and thus enhances the overall gene editing efficiency.

The SpCas9 system can recognize DNA sequences upstream of the PAM sequence 5'-NGG-3' through gRNA-directed targeting. This system can be guided by the crRNA and tracrRNA specific to the target, which can also be combined into a chimeric single-guide RNA (sgRNA). In January 2013, two papers published in Science introduced a new method for gene editing in mammalian cells based on the Streptococcus CRISPR/Cas9 system. This method used a single sgRNA to mimic the mature crRNA-tracrRNA complex, working with Cas9 to cut the target genome, marking the beginning of the widespread application of this technology.

Specification

Components

Transportation Conditions

• Cas9 Protein, T7E1 Mutation Detection Kit: Dry Ice
• Other Components: Ice Packs

Storage Conditions

• RNP Transmate Transfection Reagent: 2-8°C, do not freeze!
• Other Reagents: Store at -20°C, avoid repeated freeze-thaw cycles. For long-term use, aliquot and store at -80°C.

Principle

Whether using CRISPR/Cas9, TALENs, or ZFNs, the fundamental principle of these technologies is to induce double-strand breaks at specific genomic sequences. This allows for gene editing through the cell's own repair mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HDR). CRISPR/Cas9 gene editing technology can achieve gene knockout, gene insertion, gene modification, point mutations, and many other applications beyond gene editing.

Note: This kit is primarily designed for single sgRNA and dual sgRNA knockout applications.

CRISPR/Cas9 Gene Editing (KO) Process

Identify Target Gene
To perform gene editing on the genome, first identify the gene ID.

sgRNA Target Design
Design sgRNA targets based on the experimental objectives (for recognizing and cutting the target site). We provide free sgRNA target design services. Customers can also design targets independently. Common design websites include CRISPOR, CHOPCHOP, and E-CRISP.

Choose Gene Editing Method
CRISPR/Cas9 gene editing requires the presence of a Cas9 protein/sgRNA ribonucleoprotein complex (Cas9 RNPs) within the cell nucleus. Depending on the requirements, chemical transfection or electroporation methods can be used to introduce the complex into cells.

sgRNA and Cas9 Protein (RNP) Transfection Methods

Genotyping (KO) Methods

After introducing CRISPR components into cells, it is essential to confirm the gene editing effects by performing genotyping. The entire process includes extracting the cell genome, PCR of the target gene segments, T7E1 mutation detection, sequencing, and TIDE analysis to determine the efficiency and nature of the genetic mutations.

Indel Mutations Caused by Single sgRNA/Cas9 (NHEJ Repair):
Detection involves extracting the cell/tissue genome, performing PCR, T7E1 mutation detection, sequencing, and TIDE analysis. Since single sgRNA/Cas9-induced gene mutations typically involve small insertions or deletions (generally within 20 bp), PCR alone cannot determine whether a mutation has occurred. T7E1 endonuclease, which cuts mismatched DNA, is used to identify mutations. After denaturing and reannealing the PCR products, any indel mutations at the target site will create mismatched DNA structures flanking the target. T7E1 will recognize and cut these mismatched sites. Mutation efficiency is calculated by comparing the density of cut versus uncut bands. Sequencing of PCR products can further confirm the mutation type and efficiency.

Deletion Mutations Caused by Dual sgRNA/Cas9:
Detection involves extracting the cell/tissue genome, PCR, T7E1 mutation detection (optional), sequencing, and TIDE analysis. PCR of the target gene segment can confirm the editing efficiency of deletion fragments. The specific deletion sequence is confirmed through sequencing.

SBS Genetech is recognized as one of the global major leading industry players in Gene Editing by third-party market researchers. For more details, please visit Global Gene Editing Service Market 2019 by Company, Regions, Type and Application, Forecast to 2024.