GenWand™ Double-Stranded DNA

Pricing

dsDNA

Starting at 10 µg/item. Delivered in as fast as 3 weeks.

• Interested in getting a quote? Click the ‘Quote/Order’ button below for instant pricing.
• Already have a dsDNA-encoding plasmid stored with us? Enjoy a a 15% discount on your dsDNA order.
• For >10 kb sequence inquiries, please contact us at crispr@genscript.com .

QC Services

GenScript also offers full cGMP and IND-enabling ssDNA, dsDNA, and GenCircle™ services, accelerating your transition to the clinic!

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

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All-encompassing non-viral solutions

RUO to cGMP linear ssDNA, dsDNA and miniaturized circular dsDNA

More about dsDNA for CRISPR knock-ins

CRISPR based gene insertion, replacement, or correction

Mechanism of CRISPR HDR based gene editing

CRISPR/Cas9 is currently the most widely used to system for gene editing due to its simplicity, efficiency, precision, and versatility. In this system, the guide RNA (gRNA) recognizes the protospacer adjacent motif (PAM) sequence on the target DNA. Upon forming a complex with Cas9, the enzyme exerts its endonuclease function to create a double-stranded break (DSB). This triggers one of two primary mechanisms for repair: non-homologous end-joining (NHEJ), which introduces mutations at the DSB site, or homology-directed repair (HDR), which enables the insertion of donor DNA at the break site to achieve gene knock-in.

Double-stranded DNA (dsDNA) has long been a standard choice for HDR donor DNA templates in CRISPR knock-in applications. However, studies show that closed-end dsDNA offers distinct advantages over traditional open-end dsDNA. Closed-end structures reduce exonuclease degradation and improve stability, leading to higher editing efficiency and lower rates of off-target integration. This format is particularly ideal for knocking in long or complex sequences, and the process used to produce GenWand™ dsDNA also makes it advantageous for conducting large-scale screening or scale up.

Resources

HDR Templates Flyer

Free Download

• GenWand dsDNA outperforms PCR products in knocking in larger genes

  GenWand™ dsDNA demonstrated 80% higher KI efficiency compare to in-house PCR dsDNA (38% vs 21%)

  

  GFP KI at RAB11A Locus in HEK293 cells using electroporation with 5ug of PCR dsDNA and GenWand™ dsDNA templates.

• KI protocols

  CRISPR Knock-in Protocol for HEK 293T/293 cells with Thermo Fisher Neon® Transfection System

  CRISPR Knock-in Protocol for Jurkat cells with Thermo Fisher Neon® Transfection System

  CRISPR Knock-in Protocol for stimulated Human T Cells with Lonza 4D Nucleofector™ X Unit

  CRISPR Knock-in Protocol for stimulated Human T Cells with Maxcyte® Electroporation System

  Design and Preparation Guidelines for CRISPR KI HDR Templates

• Case studies

  Long hybrid ssDNA HDR templates enable high yield non-viral cell therapy manufacturing

  High Gene knock-in Efficiency and Reduced off Target Integration with single-stranded DNA HDR Template

  More CRISPR Case Studies and FAQs

FAQs

• Why should I choose GenWand dsDNA over PCR dsDNA?

  Our linear GenWand dsDNA provides the following advantages over PCR dsDNA:

  • High purity: GenWand dsDNA can achieve up to 99% purity
  • Better stability: proprietary closed-end modification provides increased stability
  • Worry-free: one-stop technical and project management support for your research
  • Enzymatic synthesis, animal-free and easy scale-up: save time and money while scaling production up to the gram level
  • Ideal for large insertions: for sequences of 2-10 kb in length
  • High knock-in efficiency: GenWand dsDNA has demonstrated better knock-in efficiency than PCR dsDNA for larger insertions (>2 kb), with no apparent reduction in cytotoxicity
• What is the process of producing linear GenWand dsDNA?

  To produce linear GenWand dsDNA, ssDNA is first produced from a 100% sequenced verified plasmid DNA template. The ssDNA is then cloned into a universal vector and replicated in E. coli to ensure the highest sequence fidelity. Last, special enzymes are added to produce high purity closed-end dsDNA.

• What is the maximum linear dsDNA length that you can produce?

  We can produce linear GenWand dsDNA up to 10 kb in length.

• What is the maximum quantity that you can provide for linear dsDNA?

  We can provide up to a gram per batch of linear GenWand dsDNA. However, the yield is dependent on the length and difficulty of the ssDNA sequence.

• What quality control tests do you provide for linear dsDNA templates?

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

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

  Reach out to a CRISPR expert at crispr@genscript.com if you're interested in learning about quality control for our cGMP and GMP-like GenWand dsDNA templates.

• Why do you perform double-sequencing, including the final linear dsDNA product?

  During the production of dsDNA, we do two rounds of sequencing to guarantee the sequence accuracy. We first pick a sequence-verified plasmid DNA template via sequencing to ensure the purity and sequence accuracy of the final dsDNA product. In addition, we use direct sequencing on the final dsDNA product to confirm the dsDNA product homogeneity.

• How do you purify the final linear dsDNA product?

  Column chromatography is used to purify the final linear dsDNA product upon restriction enzyme digestion.

• What delivery formats are available for GenWand dsDNA?

  Linear GenWand dsDNA can be delivered in single tubes or 96-well plates, either as dry powder or suspended in TE buffer or nuclease-free water at the concentration of your choice (up to 3mg/ml).

• What are the storage requirements for your linear dsDNA?

  GenWand dsDNA are stable for at least six (6) months. Please store at -20 °C and avoid repeated freeze and thaw cycles. Avoid repeated freeze-thaw cycles after dissolving as well. If necessary, divide the stock solution into small aliquots and only thaw the amount needed for each test.

  For more information, please refer to the GenWand dsDNA User Manual and our KI protocols.

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

  The quantity of linear GenWand dsDNA used for each test will depend on your specific application. For ex-vivo cell editing, we typically use 2-4 ug/million cells in our protocol.

• 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 is a CTS (Cas Targeting Sequence)? Should I add one to my linear dsDNA design?

  A CTS sequence is designed to allow the HDR template to interact with the sgRNA/Cas9 RNP to increase delivery of the template to the nucleus. While it is recommended to add a CTS to the 5'-end of ssDNA HDR templates, this is not the case for dsDNA templates. It has been observed that exogenous DNA (including linear dsDNA templates) can be cytotoxic at high doses and its enhanced delivery can reduce cell viability.¹

  1 https://www.semanticscholar.org/paper/Polymer-stabilized-Cas9-nanoparticles-and-modified-Nguyen-Roth/dc6ea29f77ed548e76526337e5dbae22425a5804

• Do you offer protocols for knock-ins with your HDR templates?

  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 Neon® Transfection System and MaxCyte® Electroporation Platform.

  You can view and download them for free here.

• Do you offer cGMP grade linear dsDNA?

  Yes, we proudly offer cGMP (and GMP-like) grade templates in our GenExact ssDNA and GenWand linear dsDNA formats. These services allow you to seamlessly advance your cell or gene therapy program to IND filing and clinical trials faster and more efficiently through a single vendor. Learn more about our cGMP services here.