CRISPR-Cas9 is a precise, efficient, cost-effective and easy-to-operate gene editing technology. Compared with other methods, it has a wider range of applications. Moreover, CRISPR-Cas9 technology can be used to simultaneously edit multiple genes, making it highly suitable for studying biological systems and having the potential to treat diseases by correcting defective genes. The mechanism of CRISPR-Cas9 includes the three stages of target recognition, DNA cleavage, and repair. The guide RNA (gRNA) binds to the target DNA sequence, guiding the Cas9 protein to its precise location. The gRNA and Cas9 protein form a complex to scan the DNA for the protospacer adjacent motif (PAM). After finding the PAM, the Cas9 protein cleaves the DNA at a specific location, causing a double-strand break. Subsequently, the DNA is repaired through either error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways.
CRISPR gene knockout kits typically contain the components necessary for the operation of the CRISPR-Cas9 system, including gRNA specific to the target gene, Cas9 nuclease, and sometimes a donor template for HDR. The specificity and efficiency of CRISPR-mediated gene knockout largely depend on the design of the gRNA. Well-designed gRNA can precisely direct the Cas nuclease to the desired genomic locus. Targeting the same gene with multiple gRNAs can increase the possibility of achieving complete knockout. The most used Cas nucleases are Streptococcus pyogenes Cas9 (SpCas9). The high-fidelity Cas9 variant is a modified version of standard Cas9 with reduced off-target effects. The choice between standard Cas9 and high-fidelity Cas9 depends on whether off-target effects are the main issue in the experiment. If so, the high-fidelity variant is a more suitable option. CRISPR gene knockout kits are classified based on the methods of delivering Cas enzymes and gRNAs to target cells, and the main types include plasmid-based kits, lentiviral-based kits, and ribonucleoprotein (RNP) kits. RNP is activated immediately upon delivery, resulting in rapid editing, and the Cas9 and gRNAs are degraded relatively quickly, significantly reducing the risk of off-target effects. Therefore, RNP kits becomes the preferred method for applications requiring high fidelity.
Amerigo Scientific provides gene knockout kits that contain gRNAs designed using advanced bioinformatics tools, significantly increasing the efficiency of gene knockout. Our kits aim to simplify the gene editing process for the study of gene function and disease mechanisms, as well as the development of gene therapy.
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