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Cat. No.: OSSK-20 (for 20T)

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

Description

One-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. Through a one-step reaction using this kit, they can obtain 5-25 μg of sgRNA. The obtained sgRNA can be used for in vitro analysis and evaluation of sgRNA effectiveness, as well as for transfection into Cas9-expressing cells to achieve targeted gene editing.

CRISPR/Cas9 is a groundbreaking genome editing technology that is easy to manipulate and has a wide range of applications. CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive immune system found in prokaryotes that utilizes RNA-guided DNA nucleases, such as Cas9, to silence foreign phage or viral nucleic acids. This system has gradually developed into a mature gene editing technology widely used in prokaryotes and eukaryotes. This technology can achieve site-specific cleavage of genomic DNA in prokaryotic and eukaryotic organisms under the guidance of sgRNA, followed by error-prone repair or homologous recombination to generate frameshift mutations by altering or inserting sequences at the cleavage site, thereby achieving gene knockout through gene editing. The specificity of recognizing the target site is ensured by sgRNA. With the advancement of CRISPR technology, it is now not only capable of gene knockout but also various types of mutations such as point mutations and insertion mutations, which can be used for repairing harmful mutations in clinical applications. Additionally, by constructing a Cas9 mutant variant called dCas9 that lacks endonuclease activity, sgRNA can be used for transcriptional activation or repression by directly fusing or indirectly recruiting transcriptional activators or repressors to dCas9 and targeting specific genes.

sgRNA, also known as gRNA, is composed of a CRISPR RNA (crRNA) sequence of 18-20 bp complementary to the target gene sequence and a trans-activating crRNA (tracrRNA) sequence that can specifically bind to Cas9. Inside the cell, the Cas9 nuclease can specifically bind to sgRNA, and sgRNA can also specifically bind to the corresponding target gene sequence through base pairing, leading to Cas9 nuclease cleavage at the PAM (proto-spacer adjacent motif) sequence located approximately three bases upstream of the target gene site. Subsequently, during the cell's DNA repair process, there may be insertions, deletions, or replacements at the target gene site, resulting in frameshift mutations and causing loss-of-function mutations in the target gene.

One-Step sgRNA Synthesis Kit efficiently produces sgRNA targeting the desired gene through a one-step reaction. The kit provides a Cas9 Scaffold Oligo, which is complementary to a portion of the Target-specific DNA Oligo designed by the user. Under the action of DNA polymerase, this oligo is extended to obtain a double-stranded DNA template for transcription. Then, using the RNA polymerase provided in the kit, sgRNA synthesis is catalyzed. Functional sgRNA can be obtained in a 20 µl single-tube reaction within 0.5-4 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 involved. The One-Step method is simpler, requiring only one reaction step, but it produces approximately half the yield compared to the Two-Step method. On the other hand, the Two-Step method involves a slightly more complex procedure but yields approximately twice as much sgRNA per reaction.

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.
• Use RNase-free laboratory equipment strictly, such as pipette tips, centrifuge tubes, PCR tubes, etc., and strictly avoid RNase contamination during the operation.
• The following main reagents need to be prepared: 3M NaAc (pH 5.2), ethanol (anhydrous and 70%), 1:1 water-saturated phenol/chloroform mixture. 
• 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 ordinary residential areas.
• For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage

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