Knockout Vector Construction

Leveraging its proprietary EditX™ gene editing platform, EDITGENE utilizes an optimized CRISPR/Cas9 system to design tailored knockout strategies. These are customized according to gene, cell type, and experimental goals to construct knockout vectors that meet specific research objectives.

Service Details

Service Types Knockout Lentiviral Vector / Knockout Adenoviral Vector / Knockout Adeno-Associated Viral (AAV) Vector / Knockout Plasmid
Delivery Standards Plasmid map / Plasmid sequencing results / Plasmid amplification instructions / Plasmids (3 sgRNAs provided separately)
Timeline/Pricing   Consult online for details
CRISPR-Cas9 is commonly used for gene knockout, consisting of two key components: sgRNA and the Cas9 protein. Cas9 is a gRNA-guided DNA endonuclease with the ability to specifically cleave double-stranded DNA (DSB). Under the guidance of gRNA, Cas9 recognizes and binds to the target DNA sequence. Once bound, its cutting activity is triggered, leading to a DSB, which signals the cell's repair mechanisms. There are two primary repair pathways for DSBs: non-homologous end joining (NHEJ) and homology-directed repair (HDR).
 
*Non-homologous end joining (NHEJ):In the absence of a DNA template, the cell employs the NHEJ repair pathway to directly ligate the broken ends of the DNA. However, this process often introduces small insertions or deletions (indels), which can result in gene inactivation. NHEJ is frequently exploited for gene knockout.
 
*Homology-directed repair (HDR):When a DNA template is provided, the cell can use the HDR pathway to precisely insert new DNA sequences during repair. This method is commonly used to introduce specific mutations or gene fragments.
 

Knockout Strategies

  • Frameshift Knockout

     Design sgRNA targeting the 5' coding region of the gene to induce indels that are not in multiples of three, resulting in a frameshift mutation.

  • Large Fragment Deletion

     Design sgRNA to induce the deletion of large segments within the gene.

  • Fragment Deletion

    Design sgRNA to delete short segments within the gene, causing frameshift mutations.

Service Advantages

High-Activity Cas9 Plasmid
Optimized high-activity Cas9 protein ensures more efficient gene knockout.
Efficient sgRNA Design
Proprietary sgRNA design algorithm for enhanced targeting efficiency.
Diverse Knockout Plasmid Types
Capability to construct various knockout vectors, including transient , lentiviral , and adenoviral knockout plasmids.
Scientific Design
Customized Cas9 and sgRNA expression cassettes optimized based on the target cell type.

Delivery Standards

1 Plasmid map
2 Plasmid sequencing results
3 Plasmid amplification protocol
4 Plasmids (three sgRNAs provided separately)

Plasmid Map

LentiCRISPR Lentiviral Knockout Plasmid Map
 
 
 
pSpCas9 Transient Knockout Plasmid Map

Advantage and Characteristic

Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic

Genetic Reference Book

FUT8 Gene Knockout in MIA PaCa-2 and PANC-1 Cells.
Article Title: α1,6-Fucosyltransferase contributes to cell migration and proliferation as well as to cancer stemness features in pancreatic carcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an extremely malignant tumor, accounting for 90% of all pancreatic cancers. Studies have shown that abnormal glycosylation changes on the surface of cancer cells are positively correlated with tumor progression and metastasis. α1,6-fucosyltransferase (FUT8) is a key enzyme responsible for catalyzing core fucosylation, and its abnormal expression and activation in various malignant tumors are associated with multiple physiological and pathological processes. Although the role of FUT8 in other types of cancer has been observed, its specific molecular mechanisms in PDAC malignancy transformation and potential as a therapeutic target remain unclear.
Researchers used the CRISPR/Cas9 system to knock out the FUT8 gene in MIA PaCa-2 and PANC-1 cells. The migration ability of FUT8-KO cells was evaluated using Transwell migration assays and wound healing assays, while the proliferation ability was assessed using MTT assays and colony formation assays. The expression levels of cancer stem cell markers in FUT8-KO cells were also detected. The results showed that compared to wild-type cells, FUT8-KO cells exhibited significantly reduced migration ability, proliferation, and colony formation, along with decreased expression of cancer stem cell markers. FUT8 knockout cells revealed the important role of FUT8 in pancreatic cancer and indicated that FUT8 may be a potential target for pancreatic cancer treatment, providing new insights for future therapeutic strategies against PDAC.

FAQ

What host bacteria are used for vector construction in EDITGENE? What type of strains can customers use to amplify plasmid vectors?
During vector amplification, Escherichia coli (E. coli) strains are typically used. The commonly used strain for most non-recombinant vectors is DH5α, which is suitable for most applications. For recombinant vectors, such as lentiviral vectors and transposon vectors, the Stbl3 strain can be used for amplification. Stbl3 is a specialized E. coli strain derived from HB101, which has a mutation in the recombinase gene recA13, effectively suppressing recombination of long fragment terminal repeat regions and reducing the likelihood of erroneous recombination.
When selecting a vector, consider the purpose of the experiment and the type of host cells. For example, plasmid vectors are commonly used for gene expression or amplification in bacteria, while viral vectors are more suitable for gene transfer in mammalian cells. Additionally, the vector's promoter, replicon, and antibiotic selection markers should be chosen based on specific requirements.
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