CRISPR Plasmid Construction

Some of the most critical steps for successful genome editing via CRISPR/Cas9 services are in the design and construction of sgRNA, as it determines the specificity of targeting. Our technical team takes care in designing and validating sgRNAs. Only those sgRNAs with high specificity and activity are selected before moving to downstream steps.

Service Description:

a. Consult us for genes of species other than mouse and rat.

b. Other resistant genes and fluorescent reporter genes are available upon request, please consult us.

c. Different amounts of plasmid are available upon request. Endotoxin-free maxiprep plasmids can be provided.

d. We offer in vitro sgRNA analysis services, as detailed in corresponding service descriptions.

1. Confirm sgRNA targeting gene

2. Design sgRNA and send to the client for confirmation

3. Construct and detect plasmid

4. Deliver plasmid and quality report

CRISPR Activity Assay

After designing sgRNAs, a very critical step is to select high-activity sgRNAs quickly and accurately. We developed the UCA method, a highly sensitive, simple, and convenient in vitro method capable of determining sgRNA activity rapidly. UCA is also high throughput compatible. The UCA method is based on the Single Strand Annealing (SSA) mechanism (Figure 1).

After a CRISPR/Cas9 eukaryotic expression plasmid (expressing Cas9 and sgRNA) and pUCA plasmid are co-transfected into cells, the Cas9-sgRNA complex will bind and cleave the target site corresponding to the sgRNA on the pUCA plasmid. The SSA DNA repair mechanism is then triggered, and when the homologous complementary sequence of luciferase (er) forms a complete coding sequence for luciferase, this gene will be expressed. Luciferase activity is the readout for sgRNA activity; the higher the luciferase signal, the higher the sgRNA activity.

Many sgRNA designing tools now offer the capability of predicting sgRNA activity; however, great difference remains between predicted results and actual activity. It is necessary to validate sgRNA activity experimentally to ensure the success rate of subsequent experiments.

 Our data indicate that the UCA method is capable of predicting in vivo activity of sgRNA very well; this method has been cited in high-impact journals. In summary, high-activity sgRNAs validated by Biocytogen's UCA system contribute to the success of CRISPR/Cas9 gene editing.

Service Advantages

1. Highly efficient and rapid: 2 week turnaround, from sgRNA design to validation via activity assay;

2. Sensitive, accurate, and close to in vivo activity;

3. No species restriction.

Donor Plasmid Construction

Biocytogen has more than 10 years of experience, developing thousands of mouse, rat, and cell line projects. Our rapid plasmid design and constructing services are suitable for targeting vectors (ESC/EGETM-based gene editing) and transgenic vectors (Tol2 transgenesis).

1. Confirm genes of interest, design and establish gene editing strategy

2. Design plasmid sequence and send to the client for confirmation