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Cat. No.: IVTSK-20 (for 20T)

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

Description

The In Vitro sgRNA Screening Kit is developed based on CRISPR/Cas9 gene editing technology for the in vitro screening of high-efficiency sgRNAs intended for gene editing. Taking advantage of the capability of Cas9 nuclease to cleave target DNA sequences in an sgRNA-guided in vitro reaction, this kit enables simple, reliable and rapid in vitro detection of sgRNA activity to identify high-performance sgRNA candidates.

CRISPR/Cas9 is a transformative genome editing technology characterized by simple operation and versatile applications. CRISPR, the full name of which is Clustered Regularly Interspaced Short Palindromic Repeats, refers to the adaptive immune system native to prokaryotes. This defense system utilizes the RNA-directed DNA nuclease Cas9 to silence exogenous nucleic acids derived from invading phages or viruses. Derived from this innate microbial immune mechanism, CRISPR/Cas9 has been refined into a mature gene editing tool widely applied in both prokaryotes and eukaryotes. Under the guidance of sgRNA, Cas9 introduces site-specific double-strand breaks at target loci within the genomic DNA of prokaryotic and eukaryotic organisms. Subsequent error-prone repair or homologous recombination at cleavage sites introduces sequence insertions or alterations to trigger frameshift mutations, thereby accomplishing gene knockout via genome manipulation. sgRNA is responsible for conferring target recognition specificity. With continuous advancements in CRISPR technology, its functions have expanded beyond gene knockout to diverse mutagenesis approaches including point mutations and insertion mutations. Notably, this technology holds great promise for clinical applications such as the correction of pathogenic mutations. In addition, a catalytically inactive Cas9 variant known as dCas9 can be engineered to modulate gene transcription. Either by direct fusion with transcriptional activators/repressors or indirect recruitment of such regulatory factors, dCas9 enables sgRNA-targeted transcriptional activation or repression of target genes.

sgRNA (Single Guide RNA), also simplified as gRNA, comprises two core segments: an 18–20 base pair CRISPR RNA (crRNA) sequence complementary to the target gene, and a trans-activating crRNA (tracrRNA) sequence that mediates specific binding to Cas9. Inside cells, Cas9 nuclease forms a stable complex with sgRNA, while sgRNA base-pairs precisely with its complementary target DNA sequence. This ribonucleoprotein complex directs Cas9 to cleave the target DNA approximately three nucleotides upstream of the NGG protospacer adjacent motif (PAM). Cellular DNA repair pathways subsequently mediate insertions, deletions or base substitutions at the targeted cut site, which frequently induces frameshift mutations and loss-of-function alterations in the target gene. sgRNA design is constrained by sequences flanking PAM sites. Nevertheless, even target sequences satisfying all standard design criteria often produce sgRNAs with unpredictable specificity and cleavage activity. For this reason, researchers routinely design multiple distinct sgRNAs targeting a single gene of interest and perform functional screening to select optimal candidates.

This kit supplies high-activity Cas9 Nuclease. Following the provided protocol, mix in vitro-transcribed test sgRNAs with Cas9 nuclease to digest target DNA fragments in vitro. Gel electrophoresis is then used to efficiently quantify the gene editing efficiency of multiple sgRNAs. The cleavage efficiency of each sgRNA can be quantified via grayscale analysis of electrophoresis bands. Importantly, the sgRNA activity trends measured through this in vitro screening assay are highly consistent with editing efficiencies observed in subsequent in vivo cellular experiments.

This kit does not contain reagents for in vitro transcription of sgRNA. The One-Step sgRNA Synthesis Kit or Two-Step sgRNA Synthesis Kit is recommended for sgRNA in vitro transcription.

This kit provides a positive control target DNA fragment with a length of approximately 823 bp. Digestion by Cas9 together with the positive control sgRNA generates two DNA fragments of 525 bp and 298 bp, respectively.

Components

• Cas9 Nuclease (1µM)
• 10X Reaction Buffer
• Positive Control sgRNA
• Positive Control DNA (25ng/µl)
• Proteinase K
• Ultrapure Water

Precautions

• Keep the enzyme on ice or in an ice bath during use. Immediately return it to -20 °C storage once operations are completed.
• The Positive Control sgRNA is supplied as lyophilized powder. For initial use, centrifuge the tube at 12,000 g for 5 minutes at 4 °C. Fully reconstitute the powder with ultrapure water at a ratio of 10 μL water per 250 ng sgRNA, yielding a final concentration of 25 ng/μL. Aliquot appropriately and store at -80 °C; avoid repeated freeze-thaw cycles during use.
• This kit involves manipulation of gRNA and DNA, so strict RNase-free and DNase-free practices must be followed. All self-prepared reagents and consumables shall be nuclease-free. If nuclease contamination is suspected, treat materials with 0.01% DEPC solution overnight, followed by autoclaving prior to use. Disposable face masks are recommended during operations.
• To eliminate nucleases in the working environment, RNase and DNase Away (Cat. No. R0123, Beyotime) is recommended for decontaminating lab benches, equipment and other contact surfaces. Addition of RNase Inhibitor to reaction systems is advised to prevent RNA degradation.
• This product is for research use only by qualified professionals. It shall not be used for clinical diagnosis or therapy, food or pharmaceutical manufacturing, nor stored in residential premises.
• For personal safety and health, lab coats and disposable gloves must be worn throughout all experimental procedures.

Storage

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

Related: 

• In Vitro sgRNA Screening Kit
• One-Step sgRNA Synthesis Kit
• Two-Step sgRNA Synthesis Kit