GenCircle™ Double-Stranded DNA

Advantages

Safer! No Antibiotic Resistance Gene

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• No antibiotic resistance gene & antibiotic residual
• Prevent plasmid replication outside of the engineered production host strain

Simpler! Lower Cytotoxicity & Immunogenicity

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• Eliminate CpG islands and other bacterial origin sequences
• Increase stability for longer persistence

Smaller! High fidelity 429bp backbone  

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• Higher gene expression from equivalent plasmid quantity
• Achieve higher transfection efficiency

More Efficient! Increase KI/viral packaging/GOI expression efficiency

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• Increase knock-in efficiency by up to 30%
• Improve viral titer up to 3 folds
• Increase transcription levels up to 4 folds, and protein expression levels up to 39%

Service Details

* For other GOI length & quantity request, please email [email protected] or call 1-877-436-7274.

Service options include：
1. GenScript provides GOI gene synthesis service, and then provides GenCircle dsDNA preparation service.
2. Customer provides GOI in plasmid, then GenScript provides GenCircle dsDNA preparation service.

QC Standards

GenScript also offers full cGMP and GMP-like GenCircle dsDNA services enabling faster CAR-T drug development!

State-of-the-art facility

Clean suite with class A isolator in a class C background

Comprehensive QA/QC & documentation

supporting the IND filing process

Learn More

All-encompassing non-viral solutions

RUO to cGMP linear ssDNA, dsDNA and miniaturized circular dsDNA

Case Studies

• Case 1 - GenCircle™ dsDNA as knock-in template

  Result: KI efficiency increased up to 30% compared to standard plasmid

  • CRISPR KI template（ 2.5 kb insertion）
  
  • Transposon vector for KI（4.8 kb insertion）
  

• Case 2 - GenCircle™ dsDNA as viral packaging plasmid

  Result: Viral titer improve up to 3 fold compared to standard plasmid, with stable ITR.

  • Lentivirus packaging
  

  Name	Virus Titer (IFU/ml)	Antibiotic resistance gene
  pRRL-PGK-EGFP （Regular Plasmid）	1.23E+8	Significant residue (Ct value<30)
  pRRL-PGK-EGFP MF （GenCircle dsDNA）	3.81E+8	Undetectable (Ct≥35 )

  • AAV packaging
  

  Name	Virus Titer (IFU/ml)	Antibiotic resistance gene
  CMV EGFP AAV2 (Regular Plasmid)	1.12E+12	2%
  CMV EGFP MF (GenCircle dsDNA )	1.22E+12	Undetectable

• Case 3 - GenCircle™ dsDNA used as GOI nonviral vector

  Result: RNA transcription level increased up to 4 fold, with reduced immune response compared to standard plasmid.

  

• Case 4 - GenCircle™ dsDNA used as GOI nonviral vector

  Result: protein expression level increased up to 39% compare to standard plasmid.

  

  

Successfully Delivered Case

GenScript has successfully delivered multiple orders with a wide range of GOI lengths, including multiple complex or difficult-to-synthesize projects.

FAQs

• What is the maximum insert length for GenCircle dsDNA?

  While we have only tested and delivered up to 14kb to date, we don’t foresee any limitations to delivering GenCircle dsDNA up to 50 kb in length. For any insert over 14 kb, IQR would be needed.

  As we can deliver gene synthesis up to 200 kb, GenCircle dsDNA should be able to accommodate insert sequences up to 200 kb, following a technical evaluation.

• What do I need to include in my provided sequence to generate GenCircle dsDNA?

  The GenCircle dsDNA backbone does not carry any expression or integration elements, so all functional elements for downstream applications must be placed within the insert sequence (dsDNA sequence):

  • For knock-in templates: the insert should include upstream/downstream homology arm sequences and the desired KI sequence.
  • For transposon vectors: the insert should include transposon-related elements (such as ITR) and the desired knock-in sequence.
  • For transfer plasmids used for viral packaging: the insert should include ITR for AAV (or LTR for lentivirus) and the GOI sequence.
  • For DNA vaccines/mRNA preparation templates/ protein expression: the insert should include the complete sequence of the expression cassette, containing promoter (enhancer) + ORF & functional non-coding region + poly(A) signal/terminator etc.
• How is GenCircle dsDNA produced? How is the production E.coli strain transformed with GenCircle and selected, if GenCircle has no selection marker? How do you ensure that the parental plasmid and selection marker is removed?

  The production E.coli strain is transformed with a unique parental plasmid containing a kanamycin resistance gene selection marker, and GenCircle dsDNA is then generated in vivo. After transformation and selection, the parental plasmid is induced to self-cleave into one GenCircle and one redundant backbone circle. The redundant backbone circle cannot replicate and is quickly metabolized by the production E.coli cell. GenCircle replaces the residual parental plasmid due to the unique design incompatibility of the parental plasmid backbone.
• Which steps in the GenCircle dsDNA production process are antibiotic-free? Do you test for antibiotic residuals in the final product?

  Please see the workflow below to see which steps in the production process are antibiotic-free. Colony screening and negative selection for antibiotic resistance are performed prior to fermentation and maxi prep. We do not test for antibiotic residuals in the final product.
• What is the function of R6K in GenCircle dsDNA?

  The R6K origin of replication allows GenCircle dsDNA to replicate itself in its specific host E.coli, but not in other bacteria that have not been engineered.

• Do you offer animal-free production for GenCircle dsDNA?

  Yes, for Preclinical grade GenCircle orders, we offer an animal-free option at an extra charge. Additionally, customers may request an entirely animal-free production process (culture medium, enzyme, and reagent), with a provided TSE/BSE statement.

• What are the recommended electroporation conditions for GenCircle dsDNA used in KI?

  The recommended conditions for primary T cell knock-in with the Thermo Fisher Scientific Neon™ transfection system (10ul kit) are as follows:

  • Program: 1700v, 10ms, 3pulse
  • Transfection buffer: Buffer R (the buffer in Neon kit);
  • RNP: 7.5pmol Ultra eSpCas9, 22.5pmol SafeEdit-sgRNA
  • Donor: GenCircle-dsDNA: 2ug
  • cell amount/reaction: 0.5M primary T cells

  This protocol was used by the GenScript R&D department in the knock-in case study (Case 1) above to achieve ~50% KI efficiency for a 2.5 kb insertion in the TRAC locus. We highly recommend that customers optimize conditions for their specific experiment and cell type.

• When used for lentiviral packaging, what generation system is GenCircle dsDNA? How does AAV packaging work?

  When used for lentiviral packaging, GenCircle dsDNA is a 3rd generation lentivirus system, utilizing four plasmids. GenCircle dsDNA serves as the transfer plasmid encoding your insert of interest, plus three helper plasmids.

  When used for AAV packaging, GenCircle dsDNA serves as the transfer plasmid encoding your insert of interest, plus two helper plasmids.

• What delivery formats are available for GenCircle dsDNA?

  We can deliver GenCircle dsDNA in 2 ml EP tubes (default option) or 96 well plates, formatted as lyophilized powder or in a liquid solution (TE, ddH2O, Tris, PBS, or Nuclease-free water), at the concentration of your choice.

• Does GenScript own the IP for the production process of GenCircle?

  Yes. We own the IP for GenCircle™ dsDNA and the production process of GenCircle™ dsDNA, and has applied for the international patent under PCT. Customers can use GenCircle dsDNA without IP concerns in the RUO or commercialization stage.

• Does GenScript charge any licensing fees to use GenCircle dsDNA?

  We do not charge any licensing fees for RUO applications using GenCircle dsDNA.

  For any questions regarding licensing fees for clinical or commercial use, please reach out to one of our CRISPR experts at [email protected].

• What quality control tests do you provide for GenCircle dsDNA?

  We provide two primary QC tests for our RUO grade GenCircle dsDNA templates:

  • Sanger Sequencing: ensures the accuracy of the dsDNA sequence
  • gel electrophoresis: ensures purity of the final dsDNA product.

  Pre-clinical grade GenCircle dsDNA is also tested by qPCR and HCP ELISA.

• How do you purify the final GenCircle dsDNA product?

  After the GenCircle dsDNA product replaces the residual parental plasmid, the production E. Coli cells are screened until a single colony without antibiotic resistance is selected to be used as the seed for GenCircle fermentation. This proprietary purification process ensures complete removal of the parental plasmid.

• What are the storage requirements for your circular dsDNA?

  Lyophilized GenCircle dsDNA is stable under -20°C for at least one year. Liquid products need to be stored at -20°C. Repeated freezing and thawing for either format should be avoided. If necessary, divide the stock solution into small aliquots and only thaw the amount needed for each test.

• Approximately how many tests can I perform using 2 nmol of linear dsDNA?

  Generally, 1-2 μg GenCircle dsDNA are used per million cells. Further dose optimization is necessary for your specific use case.

• Do you offer test kits or a pre-optimized sequence that I can use as a control for my initial testing or optimization studies?

  Yes! We offer Knock-in Optimization Kits (KIOK) to help you effectively optimize your CAR insertion process with a pre-validated sequence, design, and protocol. By choosing this pre-designed kit, you can achieve up to 75% in cost savings compared to ordering customized synthesis services! Learn more here.

• What are the recommended electroporation conditions for HDR knock-ins using GenCircle dsDNA?

  Yes, we offer a variety of protocols that include step-by-step instructions for HDR knock-ins with some of the most commonly used electroporators, including the Thermo Fisher Scientific Neon® Transfection System and MaxCyte® Electroporation Platform.

  You can view and download them for free here.