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

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

Description

Quick Construction Kit (CD4 Enrichment) is a rapid construction kit for building mammalian expression plasmids that express Cas9 and specific guide RNA (gRNA) based on 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 the kit to construct complete plasmids for targeted gene editing. After successful plasmid construction and transfection into mammalian cells, positive cells expressing Cas9 and gRNA can be selected using the CD4 antigen, and positive cells usually exhibit the expected gene edits in the target gene.

CRISPR/Cas9 is a groundbreaking genome editing technology that is easy to operate and widely applicable. CRISPR (Clustered regularly interspaced short palindromic repeats) is an acquired immune system in prokaryotes that utilizes RNA-guided DNA endonuclease Cas9 to silence exogenous phage or viral nucleic acids. It has gradually evolved into a mature gene editing technology widely used in both prokaryotic and eukaryotic organisms. This technology allows for site-specific cleavage of genomic DNA in prokaryotes and eukaryotes at target sequences guided by gRNA. Subsequently, error-prone repair or homologous recombination can lead to frameshift mutations or the insertion of sequences at the cleavage site, thereby achieving gene knockout through gene editing. The specificity of target recognition is ensured by gRNA. With the development of CRISPR technology, it is now possible not only to achieve gene knockout but also various types of mutations such as point mutations and insertions. Particularly in clinical applications, it can be used to repair deleterious mutations, among others. Additionally, by constructing a Cas9 mutant variant, dCas9, that lacks endonuclease activity, it is possible to achieve transcriptional activation or repression of target genes using sgRNA either through direct fusion with dCas9 or indirect recruitment of transcriptional activators or repressors.

The CRISPR/Cas9 system consists of the Cas9 nuclease and gRNA complex. The gRNA, also known as sgRNA (Single guide RNA), is composed of an 18-20 bp CRISPR RNA (crRNA) sequence that is complementary to the target gene sequence and a trans-activating crRNA (tracrRNA) sequence that can specifically bind to Cas9. By base pairing with the target sequence, the gRNA guides the Cas9 nuclease to the target DNA. The PAM-interacting domain at the C-terminus of Cas9 nuclease interacts with the Proto-spacer adjacent motif (PAM) sequence, which is typically rich in G bases (5'-NGG-3'). In the presence of the HNH and RuvC domains, a double-strand break (DSB) in the DNA is generated at a position approximately three bases upstream of the PAM sequence NGG. If this DSB occurs within the cell, it can lead to insertions, deletions, or replacements at the target site during the cell's DNA repair process, potentially resulting in frameshift mutations and loss-of-function mutations in the target gene.

Quick Construction Kit (CD4 Enrichment) provides a linearized pU6-gRNA-Cas9-T2A-CD4 plasmid that enables the simultaneous expression of target gene-specific gRNA, Cas9, and CD4. CD4 is a hallmark protein of helper T cells and plays a crucial role in immune responses. It has multiple functions in both intracellular and extracellular environments when faced with disruptions. CD4 can participate in the development of helper T cells in the thymus and trigger the differentiation of monocytes into functionally mature macrophages. The antigenic properties of CD4 can be utilized for magnetic bead enrichment of cells expressing Cas9 and gRNA. Additionally, fluorescence labeling of CD4 allows for flow cytometric cell sorting and single-cell cloning of cells expressing Cas9 and gRNA.

The plasmid contains a T2A peptide sequence between the coding sequences of Cas9 and CD4. T2A is a "self-cleaving" peptide that can be understood as consisting of 18 amino acid residues (EGRGSLLTCGDVEENPGP). However, the actual process is not self-cleavage but rather the bypassing of the synthesis of the glycine and proline peptide bonds at the C-terminus of T2A and other 2A elements by the ribosome, resulting in the separation of the 2A sequence's C-terminus and downstream product. Additional T2A residues (GSGEGRGSLLTCGDVEENPG) will be added to the C-terminus of the upstream Cas9 protein, while the N-terminus of the downstream CD4 protein will have additional proline residues. Adding the GSG sequence at the N-terminus of the T2A peptide improves cleavage efficiency.

The pU6-gRNA-Cas9-T2A-CD4 plasmid confers ampicillin resistance.

The sequencing primer sequences available for the pU6-gRNA-Cas9-T2A-CD4 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 product; the ligation product can be directly used for transformation. However, when using electroporation to transform Escherichia coli, it is generally recommended to first purify the DNA using a DNA purification kit or phenol-chloroform extraction method before proceeding with electroporation.
• It is not necessary to perform agarose gel electrophoresis for regular ligation reactions. If gel electrophoresis is required for the ligation product, it is recommended to incubate at 65°C for 10 minutes to inactivate T4 DNA Ligase and prevent band shifting caused by binding of T4 DNA Ligase to DNA.
• This product is intended for scientific research use by professionals only. It is not for clinical diagnosis or treatment, and should not be used for food or drugs. It should not be stored in a regular household.
• 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)