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Cat. No.: QZE-20 (for 20T)

Cat. No.: QZE-100 (for 5*20T)

Description

The sgRNA Quick Construction Kit (Zeocin) is developed based on CRISPR/Cas9 genome-editing technology for rapid assembly of mammalian expression plasmids that produce custom guide RNA (gRNA). Users only need to design and synthesize target-specific DNA oligos following the product manual. After annealing to form double-stranded DNA fragments, the fragments are ligated into the vector supplied with this kit to generate complete expression plasmids encoding target-specific sgRNA for CRISPR experiments.

Upon successful plasmid construction, the sgRNA plasmid can be co-transfected into mammalian cells together with a Cas9 or dCas9 (dead Cas9) expression plasmid. The Zeocin resistance gene enables selection of cell populations with stable sgRNA expression. These positive cells typically exhibit the desired genome editing or transcriptional modulation of the target gene.

CRISPR/Cas9 is a revolutionary genome-editing tool featuring simple workflows and broad applicability. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) originates as an adaptive immune system in prokaryotes, wherein the RNA-guided DNA nuclease Cas9 silences foreign genetic material from invading phages and viruses. This natural defense mechanism has been engineered into a sophisticated, versatile gene-editing platform compatible with both prokaryotic and eukaryotic organisms.

Under the guidance of gRNA, Cas9 mediates site-specific cleavage of target genomic DNA in prokaryotes and eukaryotes. Cellular repair pathways—error-prone non-homologous end joining or homologous recombination—introduce sequence insertions, deletions or substitutions at the breakpoints, frequently inducing frameshift mutations and resulting in targeted gene knockout. The gRNA confers strict specificity for target locus recognition. Continuous advances in CRISPR methodology have expanded its functions beyond gene knockout to precise generation of point mutations and knock-in insertions; notably, this system holds great clinical potential for correcting pathogenic gene variants.

The CRISPR/Cas9 functional unit consists of a ribonucleoprotein complex formed by Cas9 Nuclease and gRNA. Also known as single guide RNA (sgRNA), gRNA comprises two core segments: an 18–20 bp CRISPR RNA (crRNA) stretch complementary to the target gene, and a trans-activating crRNA (tracrRNA) domain that specifically associates with Cas9.

Base pairing between gRNA and its complementary genomic target recruits Cas9 Nuclease to the target DNA locus. The C-terminal PAM-interacting domain of Cas9 Nuclease recognizes the G-rich canonical PAM motif 5′-NGG-3′. Coordinated catalytic activity of the HNH and RuvC nuclease domains generates a DNA double-strand break (DSB) approximately three nucleotides upstream of the NGG PAM sequence. Intracellular DSBs trigger endogenous DNA repair machinery, which introduces random insertions, deletions or base substitutions at the targeted site. Such lesions often trigger frameshift mutations and lead to loss-of-function knockout of the target gene.

Mutagenesis of both the HNH and RuvC domains of Cas9 Nuclease yields catalytically inactive dCas9. Although dCas9 loses DNA cleavage capacity, it still binds sgRNA and is recruited to target DNA sequences with high specificity under sgRNA guidance. Fusing dCas9 directly to transcriptional regulators or enabling it to indirectly recruit such factors enables targeted transcriptional activation or repression of sgRNA-targeted genes. When dCas9 is fused with epigenetic modifiers, targeted epigenetic modifications (such as methylation/demethylation and acetylation/deacetylation) can be introduced at the target locus to modulate target gene expression.

We also offer a full portfolio of CRISPR-Cas9 genome editing products, including Cas9 lentiviruses, stably transfected cell lines, and detection kits for gene editing, delivering a one-stop solution to accelerate customers’ genome editing workflows. Please visit our official website for detailed product information.

We additionally provide CRISPR-dCas9-based plasmid systems dedicated to gene expression modulation, such as transcriptional regulation and epigenetic modification, available for separate purchase based on your research demands. Construct sgRNA expression plasmids targeting your gene of interest using the vectors supplied in this kit, then co-transfect them with the aforementioned dCas9 regulatory plasmids to achieve precise transcriptional control over target genes. For more details, browse the Beyotime official website or click the related product links on this product page.

The pU6-gRNA vector provided in this kit carries two recognition sites for the Type IIS restriction enzyme BsaI, enabling rapid sgRNA plasmid construction via Golden Gate Assembly. This vector simultaneously expresses sgRNA targeting mammalian genes of interest and the Zeocin resistance gene (Bleo resistance). Zeocin selection can be applied to isolate polyclonal or monoclonal cell populations stably expressing sgRNA. sgRNA expression plasmids constructed with this kit are compatible with CRISPR-Cas9-mediated genome editing and gene expression regulation assays.

The pU6-gRNA plasmid confers kanamycin resistance for bacterial selection and bleomycin resistance for mammalian cell screening. It confers kanamycin resistance when propagated in E. coli. After transfection into mammalian cells, Zeocin (bleomycin) can be used to select polyclonal or monoclonal cell lines with stable sgRNA expression.

This kit supplies the sequencing primer for the pU6-gRNA plasmid (hU6-F Primer, 10 µM). The recommended sequencing primer sequence is listed below:

hU6-F Primer: 5′-GAGGGCCTATTTCCCATGATT-3′

Precautions

• For regular transformation of Escherichia coli, there is no need to purify the ligation product. The ligation product can be directly used for transformation. However, when using electroporation for the transformation of Escherichia coli, it is generally recommended to purify the DNA using a DNA purification kit or methods such as phenol-chloroform extraction before electroporation.
• It is not necessary to perform gel electrophoresis for regular ligation reactions. If gel electrophoresis is required for observing the ligation product, it is recommended to incubate the reaction at 65°C for 10 minutes to inactivate T4 DNA Ligase, to avoid band shifting caused by the binding of T4 DNA Ligase to DNA.
• This product is intended for scientific research purposes by professionals only. It is not intended for clinical diagnosis or treatment, and should not be used for food or drugs. It should not be stored in a regular residential area.
• For your safety and health, please wear appropriate laboratory attire and disposable gloves during the operation.

Storage

The minimum shelf life is 2 years at -20°C.

Related: 

• sgRNA Quick Construction Kit (Zeocin)
• Quick Construction Kit (Puro)
• Quick Construction Kit (CD4 Enrichment)
• Quick Construction Kit (mOrange2 Reporter)