Custom Single Guide RNA (sgRNA) Synthesis

CD Formulation offers sgRNA design and chemical synthesis services. Our products undergo mass spectrometry testing to verify the accuracy of each sequence. We also provide HPLC purification and ensure batch-to-batch stability, delivering high-quality sgRNA to our clients.

Background of Single Guide RNA (sgRNA) Synthesis

sgRNA (single guide RNA) serves as the of the CRISPR/Cas9 gene editing system, guiding Cas9 to perform targeted DNA editing. Chemically synthesized sgRNAs typically exhibit low cytotoxicity, and their stability can be enhanced through various modifications to minimize off-target effects. They offer advantages over in vitro transcribed and plasmid-based sgRNAs, including lower cost, higher editing efficiency, and improved consistency. Additionally, gRNAs consist of tracrRNA and at least one crRNA. The term refers to the precise design of the crRNA components to select the most suitable sgRNA.

Principles of sgRNA Design

• Ensure that the sgRNA is specific to prevent non-specific editing in the genome. Avoid selecting regions that are similar to other gene sequences, particularly those with multiple copies in the genome.
• sgRNAs should have a GC content between 40% and 60% to ensure optimal stability and hybridization under experimental conditions.
• Select sgRNAs with a single target shear site to prevent unintended multiple gene edits.
• Refrain from selecting regions in the genome that contain repetitive or highly similar sequences to minimize the risk of non-specific shearing.
• Ensure that sgRNA sequences do not contain stable secondary structures to enhance their effectiveness under experimental conditions.
• Avoid selecting sgRNAs that match multiple sites in the genome. sgRNAs with a high rate of shifted mutations and fewer off-target sites can be evaluated using sgRNA design software.
• When constructing T7/U6 promoter-driven sgRNA expression vectors, it is important to ensure that the 5' base of the sgRNA is preferably a guanine (G). If it is not, a guanine can be artificially added to enhance transcription efficiency.
• To induce a code-shift mutation, the sgRNA should be designed within the coding sequence (CDS) region, preferably near the N-terminus of the protein. It is ideal for the sgRNA to be located in the first or second exon to produce a non-functional protein.
• When multiple transcripts of a gene are present, it is advisable to design the sgRNA in the homologous region.

Explore Our Custom Single Guide RNA (sgRNA) Synthesis Services

sgRNA Design

sgRNA design is essential in CRISPR technology, focusing on selecting specific target gene regions for effective gene editing. Important considerations include minimizing off-target effects, PAM sequence positioning, GC content, and secondary structure, all of which enhance the precision and efficiency of targeted DNA cleavage.

Construction of sgRNA Plasmids

For the construction of sgRNA plasmids, designed sgRNA sequences are cloned into appropriate plasmid vectors to facilitate the expression of the desired sgRNAs. The plasmid must be validated using methods such as DNA sequencing to ensure that it can effectively instruct the CRISPR-associated nuclease to edit the target gene.

Single Guide RNA (sgRNA) Customization

CD Formulation offers chemically synthesized, high-quality single guide RNA (sgRNA), tracrRNA, and crRNA to enhance your gene editing experience. We can provide sgRNA with the following characteristics:

• The synthesized length is between 93 and 103 nucleotides.
• Quality control in mass spectrometry to ensure 100% accurate sequencing.
• The results are consistent across batches.
• Lower toxicity, increased stability, and high editing efficiency make it an excellent choice for enhancing gene editing effectiveness.

General Workflow for Single Guide RNA (sgRNA) Preparation

Fig.1 Flow chart of sgRNA preparation. (CD Formulation)

• Sequence Design

Identify the target gene and design the corresponding sgRNA sequence. Ensure the specificity and efficiency of the sgRNA sequence.

• DNA Template Synthesis

Synthesize the DNA template for sgRNA via chemical synthesis or PCR amplification. The template sequence usually includes the T7 promoter and the sgRNA sequence.

• In Vitro Transcription

Use T7 RNA polymerase to transcribe the synthesized DNA template in vitro into RNA sequences to produce sgRNA.

• sgRNA Purification

Purify the transcribed sgRNA using electrophoresis or column chromatography to remove impurities and untranscribed template DNA from the reaction.

• Quality Analysis

Quality control for the purified sgRNA usually involves using methods like gel electrophoresis or mass spectrometry. These techniques help ensure the sgRNA's integrity and purity.

• sgRNA Storage and Usage

Dissolve the final high-purity sgRNA in an appropriate buffer solution for storage and proceed with subsequent usage and testing based on experimental needs.

Our sgRNA Synthesis Technology Platforms

CD Formulation can utilize a chemical synthesis technology platform to produce sgRNA efficiently by systematically synthesizing the sgRNA building blocks and assembling them into the complete sgRNA structure.

Advantages of the chemical synthesis technology platform:

• Efficiency
• Precision
• Scalability
• Flexibility
• High purity
• Cost-effective

Why Choose Us for Customized sgRNA Synthesis?

• High Synthesis Yield - Yields of 20 to 100 μg of sgRNA can be obtained in a single tube within 4 hours.
• Wide Range of Applications - High yields of various types of sgRNA can be achieved.
• High Purity - The synthesized sgRNA exhibits a single band.
• High Activity - Synthesized sgRNA can effectively direct Cas9 to cleave target DNA.

Publication Data

CD Formulation, leveraging extensive experience in nucleic acid synthesis and supported by a team of skilled technicians, offers high-quality, multi-species nucleic acid synthesis services delivered promptly. We ensure the quality of our products meets customer requirements by implementing a rigorous supplier quality management system, maintaining strict control over intermediate production processes, and selecting safe and efficient logistics services. Contact us for a tailored solution for your project.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.