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Cat. No.: TSSK-20 (for 20T)

Cat. No.: TSSK-100 (for 100T)

Description

Two-Step sgRNA Synthesis Kit is a reagent kit developed for the in vitro transcription synthesis of sgRNA (single guide RNA) based on CRISPR/Cas9 gene editing technology. With this kit, users only need to design and synthesize target-specific DNA oligos according to the kit's instructions, and they can obtain 10-40 μg of sgRNA per reaction. The obtained sgRNA can be used for in vitro analysis and assessment of sgRNA efficacy, as well as for transfection into Cas9-expressing cells to achieve gene editing of the target gene.

CRISPR/Cas9 is a groundbreaking genome editing technology that is easy to manipulate and widely applicable. CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive immune system found in prokaryotes that utilizes RNA-guided DNA endonuclease Cas9 to silence exogenous bacteriophage or viral nucleic acids. This system has gradually evolved into a mature gene editing technology widely used in prokaryotic and eukaryotic organisms. The technique allows for site-specific cleavage of the genomic DNA of prokaryotic and eukaryotic organisms under the guidance of sgRNA through Cas9. This is followed by error-prone repair or homologous recombination to induce frame-shifting mutations or introduce sequence insertions at the cleavage site, thereby achieving gene editing and gene knockout. The specificity of target recognition is ensured by sgRNA. With the development of CRISPR technology, it is now not only capable of gene knockout but also various types of mutations such as point mutations and insertions. It is particularly useful in clinical applications for repairing detrimental mutations [1, 2]. Additionally, by constructing a Cas9 mutant variant, dCas9, which lacks endonuclease activity, and directly fusing it with dCas9 or indirectly recruiting transcription activation or repression factors, sgRNA-mediated transcription activation or repression of target genes can be achieved.

sgRNA, also known as gRNA, consists of a CRISPR RNA (crRNA) sequence complementary to the target gene sequence, ranging from 18 to 20 base pairs, and a trans-activating CRISPR RNA (tracrRNA) sequence that can specifically bind to Cas9. In cells, the Cas9 nuclease can specifically bind to sgRNA, and sgRNA can also specifically bind to the corresponding target gene sequence through base pairing. This results in the Cas9 nuclease cleaving the target gene at the PAM (proto-spacer adjacent motif) sequence, which is located approximately three bases upstream of the NGG sequence. Subsequently, during the cellular DNA repair process, insertions, deletions, or replacements can occur at the gene target site, potentially resulting in frame-shift mutations and loss-of-function mutations in the target gene.

Two-Step sgRNA Synthesis Kit efficiently produces target-specific sgRNA for the desired gene through a two-step process involving PCR amplification and transcription dependent on T7 RNA polymerase. The kit provides a pre-mixed Scaffold Template, which includes an 80-nt sequence that overlaps with the user-designed target-specific sequence, as well as primers for amplifying this sequence. After annealing to form a complementary strand, the DNA polymerase extends the sequence, ultimately generating a double-stranded DNA template for transcription. Using the reagents provided in the kit and the user's own designed specific DNA sequence, 10-40 μg of functional sgRNA can be obtained per reaction in a single tube reaction within 0.5 to 4 hours. Purified sgRNA can be transfected into cells for gene editing in Cas9-expressing cells or mixed with Cas9 nuclease for enzyme cleavage identification experiments.

The main difference between the One-Step sgRNA Synthesis Kit and the Two-Step sgRNA Synthesis Kit lies in the process. The One-Step kit offers a simple one-step reaction, while the Two-Step kit involves a slightly more complex two-step process. However, the yield of the One-Step kit is approximately half that of the Two-Step kit. In other words, each reaction using the Two-Step kit produces approximately twice the amount of sgRNA compared to the One-Step kit.

Precautions

• The enzyme should be stored in an icebox or on an ice bath during use and immediately placed at -20°C after use for storage.
• Strictly use RNase-free laboratory equipment (such as pipette tips, centrifuge tubes, PCR tubes, etc.), and take strict precautions to avoid RNase contamination during the operation.
• The main reagents to be prepared by yourself: 3M NaAc (pH 5.2); absolute ethanol; 70% ethanol; 1:1 mixture of saturated phenol/water-saturated chloroform.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in a regular residential setting.
• For your safety and health, please wear laboratory attire and disposable gloves when handling.

Storage

The minimum shelf life is 1 year at -20°C.