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The General Introduction of sgRNA Synthesis

CRISPR/Cas9 gene editing technology has revolutionized research across various fields, dramatically enhancing the efficiency of genome editing. Unlike traditional methods the direct delivery of ribonucleoprotein (RNP) complexes, formed by Cas9 and sgRNA, into cells is faster, less immunogenic, and has lower off-target effects while achieving higher editing efficiency.

At Synbio Technologies, we offer expert synthesis services for gene editing researchers. Synbio Technologies provides two sgRNA synthesis strategies: chemical synthesis and in vitro transcription.

Highlights

Ready-to-use sgRNA Products
Complete Modification System
Batch-to-batch Consistency and Traceability
Rnase-free with Strict QC
Highly Customizable Services

Service Details

• Chemically Synthesized sgRNA

Synbio Technologies delivers 100% accurate sgRNA sequences with desired chemical modifications. These sgRNAs offer high stability and low toxicity, ensuring high editing efficiency. These products are also easy to use and require no annealing.

Length (nt)	Yield	Modification	Purification	Price	Turnaround Time
95-105 nt	1-10 OD	2’-OMe and Phosphorothioate	HPLC	Quote	7-10 BD

• In Vitro Transcribed sgRNA

We provide a one-stop solution from design to synthesis of sgRNAs. Synbio Technologies has delivered sgRNAs that have been used successfully in zebrafish, mice, filamentous fungi, and various other species. These sgRNAs are delivered quickly, with up to 20 μg yields in as soon as 3 days, including ready for direct injection or transfection for gene editing experiments.

Service	Service details
IVT sgRNA	sgRNA design DNA template synthesis In vitro transcription and purification

QUOTE

Deliverables

Lyophilized RNA

Certificate of Analysis (COA)

Case Study

Our sgRNA Design Center designed 8 sgRNAs per gene for multiple genes in mice and conducted in vitro transcription within 2 days, producing 10-20 μg sgRNAs for rapid use in cytological experiments.

Experimental Procedures:

One-step synthesis of gRNA DNA template

Experimental Results:

1. The gRNA DNA template was cloned into the pUC57 vector using blunt-end ligated and verified with Sanger sequencing. The comparison results were consistent with the designed sequence. As shown in Figure 1.

Figure 1. Plot of blunt-end ligated results against the design sequence

2. Electrophoresis in 2% agarose was performed on the sgRNA obtained by in vitro transcription and clear bands could be seen. This is shown in Figure 2.

Figure 2. sgRNA agarose gel electropherogram

3.One sgRNA sequence was verified: The selected sgRNA was reverse transcribed to obtain cDNA, sgRNA amplification primers were designed, and the DNA sequence of sgRNA was obtained through PCR. Then the DNA sequence was cloned into the pUC57 vector using blunt-end ligation and verified with Sanger sequencing. The resulting sequencing data verified that the sgRNA sequence was correct. This is shown in Figure 3.

Figure 3. sgRNA sequence validation agarose gel electrophoretogram

* Lane1: sgRNA reverse transcribed cDNA; Lane2: DNaseI treated sgRNA; Lane3: DNA template for in vitro transcription

FAQs

How can off-target effects of sgRNA be avoided?

- Using highly specific Cas9 variants.

- Precise design of guide sequences to minimize similarity to non-target sequences.

- Use bioinformatics tools to predict potential off-target sites and optimize them when designing sgRNAs.

How to improve the stability and activity of sgRNA?

The stability and activity of sgRNAs can be improved by chemical modifications (e.g., at the 2'-OH site). In addition, optimizing the design of sgRNAs to reduce off-target effects can also help improve their efficiency.

what things are important to consider when designing a sgRNA?

When designing a sgRNA, consider the target sequence specificity and ensure the presence of a suitable PAM sequence near the target site to minimize off-target effects. Additionally, stabilize the GC content, avoid strong secondary structures, and validate the sgRNA's efficiency and specificity experimentally.

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