All products have special prices for bulk purchase, please contact for more details if required.

Cat. No.: QMOR-10 (for 10T)

Cat. No.: QMOR-50 (for 50T)

Description

Quick Construction Kit (mOrange2 Reporter) is a rapid construction kit for mammalian expression plasmids used in the construction of Cas9 and specific guide RNA (gRNA) for gene editing based on the CRISPR/Cas9 gene editing technology. Users only need to design and synthesize target-specific DNA oligos according to the instructions, anneal them to form double-stranded DNA, and then ligate them into the linearized vector provided in this kit to construct a complete plasmid for targeted gene editing. Once the plasmid is successfully constructed and transfected into mammalian cells, positive cells expressing Cas9 and gRNA can be screened by the orange-red fluorescence of mOrange2. Typically, the desired gene editing of the target gene will be observed in the positive cells.

CRISPR/Cas9 is a revolutionary genome editing technology that is easy to manipulate and widely applicable. CRISPR (Clustered regularly interspaced short palindromic repeats) is an acquired immune system found in prokaryotes, where the RNA-guided DNA endonuclease Cas9 targets and silences foreign phage or viral nucleic acids. Over time, this system has been developed into a mature gene editing technology widely used in both prokaryotic and eukaryotic organisms. The CRISPR/Cas9 technology allows for site-specific cleavage of the genomic DNA in prokaryotic and eukaryotic organisms at targeted sequences guided by the gRNA. Subsequently, error-prone repair or homologous recombination can introduce insertions or changes at the cleavage site, leading to frameshift mutations and gene knockout through gene editing. The specificity of target recognition is ensured by the gRNA. With the advancement of CRISPR technology, it is not only possible to achieve gene knockout but also various types of mutations such as point mutations and insertions. In clinical applications, it can be used to repair deleterious mutations and more. Additionally, by constructing a Cas9 mutant variant, dCas9, which lacks endonuclease activity, it can be directly fused with transcriptional activation or repression factors or recruited to achieve transcriptional activation or repression of target genes using specific gRNAs.

The CRISPR/Cas9 system consists of the Cas9 nuclease and the gRNA complex. The gRNA, also known as single guide RNA (sgRNA), is composed of a CRISPR RNA (crRNA) sequence complementary to the target gene sequence of 18-20 base pairs and a trans-activating crRNA (tracrRNA) sequence that can bind specifically to Cas9. By complementarily pairing with the target sequence, the gRNA guides the Cas9 nuclease to the target DNA. The PAM-interacting domain at the C-terminal of Cas9 nuclease recognizes the PAM sequence (5'-NGG-3'), which is rich in G bases, and in collaboration with the HNH and RuvC domains, induces a double-strand break (DSB) in the DNA at approximately three bases upstream of the PAM sequence NGG. If this DSB occurs within a cell, the repair process can lead to insertions, deletions, or replacements at the target site, potentially resulting in frameshift mutations and the loss of the target gene's function.

The provided kit contains the linearized pU6-gRNA-Cas9-T2A-mOrange2 plasmid, which simultaneously expresses the gRNA targeting the desired gene, Cas9 nuclease, and the fluorescent protein mOrange2. mOrange2 is an extremely bright orange-red fluorescent protein with higher protein stability compared to mOrange. It can be used for flow cytometry-based cell sorting or single-cell cloning of cells expressing Cas9 and gRNA. The plasmid contains a T2A peptide sequence between the coding sequences of Cas9 and mOrange2. T2A can be understood as a "self-cleaving" peptide with 18 amino acid residues (EGRGSLLTCGDVEENPGP), but the actual process does not involve self-cleavage. Instead, it allows the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminal of the 2A element, resulting in the separation of the 2A sequence from the downstream product. The C-terminal of the upstream Cas9 protein will have additional T2A residues (GSGEGRGSLLTCGDVEENPG), while the N-terminal of the downstream mOrange2 protein will have an additional proline residue. Adding a GSG sequence at the N-terminal of the T2A peptide can enhance cleavage efficiency.

The pU6-gRNA-Cas9-T2A-mOrange2 plasmid confers ampicillin resistance.

The sequencing primer sequences for the pU6-gRNA-Cas9-T2A-mOrange2 plasmid are as follows:

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

Precautions

• For regular transformation of Escherichia coli, there is no need to purify the ligation products. The ligation products can be directly used for transformation. However, when using electroporation for E. coli transformation, it is recommended to first purify the DNA using a DNA purification kit or phenol-chloroform extraction method before proceeding with electroporation.
• Routine ligation reactions do not require gel electrophoresis analysis. If gel electrophoresis analysis is necessary for the ligation products, it is recommended to incubate the reaction at 65°C for 10 minutes to inactivate T4 DNA Ligase and prevent band shift caused by the interaction between T4 DNA Ligase and DNA.
• This product is intended for use by professionals in scientific research and is not intended for clinical diagnosis or treatment, food or drug use, or storage in regular residential areas.
• For your safety and health, please wear appropriate laboratory attire and disposable gloves when handling.

Storage

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

Related: Quick Construction Kit (Puro), Quick Construction Kit (CD4 Enrichment), Quick Construction Kit (mOrange2 Reporter)