Other Services

CRISPR Plasmid Construction

Critical steps in successful genome editing via CRISPR/Cas9 include the design and construction of sgRNA, as it determines targeting specificity. 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

sgRNA Targeting Species sgRNA Promoter Plasmid Screening Markersb Deliverables Delivery Criteriac Optional Servicesd Delivery Time
Rat, Mouse, Human, Primate, Other Mammals. a U6

T7

PuroR

mCherry

Plasmid

Sequencing file

Quality report

Total amount > 5 μg

Concentration > 50 ng/μL

Enzyme digestion validation

Sanger sequencing

sgRNA efficiency determination 10 business days
  1. Consult us for genes of species other than mouse and rat.
  2. Other resistant genes and fluorescent reporter genes are available upon request, please consult us.
  3. Different amounts of plasmid are available upon request. Endotoxin-free maxiprep plasmid can be provided.
  4. We offer in vitro sgRNA analysis services, as detailed in corresponding service descriptions.

Service Procedure

  • Confirm sgRNA targeting gene
  • Design sgRNA and send to the client for confirmation
  • Construct and detect plasmid
  • Delivery plasmid and quality report

CRISPR Activity Assay

After designing sgRNAs in silico, a very critical step is to select high-activity sgRNAs quickly and accurately. We developed the UCA method, [YL1], 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 [1] (Figure 1).

technology-UCA-method

 

Figure 1. Schematic diagram of CRISPR/Cas9 activity determination using SSA mechanism.

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 [2,3]. 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

[1]. Mashiko, D, et al. “Feasibility for large-scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. ” Development Growth & Differentiation 56.1(2014):122.

[2]. Wu M, Wei C, Lian Z, et al. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system[J]. Scientific reports, 2016, 6.

[3]. Lin, Zhimiao, et al. “Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility.” Nature Genetics 48.12 (2016): 1508-1516.

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).

Service Description

Speciesa Deliverables Delivery Criteria Optional Services
 Rat

Mouse

Cell line

 Plasmid

Sequencing file

Quality report

 Total amount > 5 μg

Concentration > 50 ng/μL

Enzyme digestion validation

Sanger sequencing

Endotoxin-free maxiprep plasmid

Pronucleus injection plasmid preparation

Service Procedure

  1. Confirm genes of interest, design and establish gene editing strategy;
  2. Design plasmid sequence and send to the client for confirmation;
  3. Construct the plasmid;
  4. Deliver plasmid. 
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