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

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

Description

Quick Construction Kit (Puro) is a rapid construction kit for mammalian expression plasmids designed for the expression of Cas9 and specific guide RNA (gRNA) using CRISPR/Cas9 gene editing technology. Users only need to design and synthesize target-specific DNA oligos according to the instructions, and after annealing to form a double-stranded DNA, they can be ligated 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 selected using the Puro resistance gene, and these 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.

The linearized pU6-gRNA-Cas9-T2A-Puro provided in this kit enables the simultaneous expression of target-specific gRNA, Cas9, and the puromycin resistance gene (PuroR). Puromycin Dihydrochloride (Puromycin) can be used for selection of multiclonal or monoclonal cells expressing Cas9 and gRNA using the puromycin resistance. Puromycin is known for its rapid action on cells, typically killing 99% of cells that do not express the PuroR gene within 2 days. The plasmid contains a T2A peptide sequence between the coding sequences of Cas9 and Puro. T2A is a "self-cleaving" peptide that can be understood as containing 18 amino acid residues (EGRGSLLTCGDVEENPGP). However, the actual process does not involve self-cleavage but rather allows the ribosome to bypass the synthesis of the glycine and proline peptide bonds at the C-terminus of the T2A element, leading to separation of the 2A sequence's C-terminus from the downstream product. The upstream Cas9 protein will have additional T2A residues (GSGEGRGSLLTCGDVEENPG), while the downstream Puro protein will have an additional proline residue at its N-terminus. Adding a GSG sequence at the N-terminus of the T2A peptide improves cleavage efficiency.

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

The sequencing primer sequences that can be used for the pU6-gRNA-Cas9-T2A-Puro 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 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: Quick Construction Kit (Puro), Quick Construction Kit (CD4 Enrichment), Quick Construction Kit (mOrange2 Reporter)