GenExact™ Single-Stranded DNA

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• Interested in getting a quote for your specific sequence? Click the ‘Quote/Order’ button below for instant pricing.
• Already have an ssDNA-encoding plasmid stored with us? Enjoy a 15% discount on your ssDNA order.
• For >100 µg or >5 kb sequence inquiries, please contact us at crispr@genscript.com.

* We also offer <200nt ssDNA through our DNA Oligo Chemical Synthesis Service.

QC Services

GenScript also offers full cGMP and IND-enabling ssDNA, linear 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 ssDNA for CRISPR knock-ins

CRISPR based gene insertion, replacement, or correction

How HDR-based CRISPR gene editing works

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 traditionally been used for HDR templates. However, recent studies have demonstrated that single-stranded DNA (ssDNA or ssODN) is the most effective HDR template for CRISPR-based gene insertion, replacement, and correction1-5. Compared to dsDNA donors, ssDNA has shown significantly improved editing efficiency and specificity, along with reduced off-target integration- especially when editing primary cells and stem cells, or developing transgenic animal models.

Related Publications

• 1."We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement." Miura H, et al., Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc. (2018) 13(1):195-215.
• 2."We report that targeted chromosomal translocations are generated more efficiently when the all-in-one plasmid, RNP complex, and ssODN-based approaches are used, with the most efficient strategy being the combination of RNP complexes with translocation-ssODNs." Torres-Ruiz, et al., Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9. Stem Cell Reports. (2017) 8: 1408–1420.
• 3."ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration." Li, et al., Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv 178905; doi: https://doi.org/10.1101/178905.
• 4."The PITCh approach required 265 zygotes whereas Easi-CRISPR used only 105 zygotes, and the PITCh approach produced 33% correctly targeted pups, whereas Easi-CRISPR (using ssDNA donors) produced 100% correctly targeted pups." Quadros, et al., Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biology (2017) 18:92.
• 5."Similar to what has been described with viral HDR templates, we found evidence to suggest that double-stranded templates could integrate independent of target homology, albeit at low rates. These rare events could be reduced almost completely by using single-stranded DNA (ssDNA) templates." Roth, et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559 (2018) 405–409.

Resources

HDR Templates Flyer

Free Download

• Highly efficient non-viral T cell engineering process at clinical scale with GenExact ssDNA

  Collaboration with Marson's lab from UCSF (University of California San Francisco) showed that:

  • GenExact™ ssDNA consistently outperformed in-house generated HDRTs (homology-directed repair templates), showing lower levels of toxicity and higher knock-in efficiencies;
  • The KI efficiency of GenExact™ ssDNA via electroporation without any enhancer can reach to 46.2% at a GMP-compatible scale;
  • KI positive live cells count generated by GenExact™ ssDNA was way above estimated patient dose (100*10e6). In vitro assays demonstrated efficient killing of BCMA-CAR cells.

  

  Brian R Shy. et.al., bioRxiv(2021), Read the whole article.

• 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

  Using Long ssDNA for the Generation of Foxi1-rtTA Transgenic Mice

  More CRISPR Case Studies and FAQs

• 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

• Featured

  CRISPR Expands CAR T Cell Possibilities

  Choosing the Right CRISPR Tools to Generate Your Genetically Modified Mice

  Get The Correct Pup in The First Round

• Webinars
  • CRISPR in Creating Knockin Cell Lines and Animal Models - Functionalizing Genome Editing for a Broad Range of Targets
  • In vivo delivery of Cas9 ribonucleoprotein and donor DNA with gold nanoparticles
  • CRISPR Based T Cell Editing: Large knock-ins in human T cells using non-viral HDR templates
  • Strategies to Efficiently Generate CRISPR KO/KI Cell Lines

FAQs

• What factors do I need to consider to design an effective ssDNA template?

  For point mutations, we suggest using an asymmetric ssDNA design. This paper from 2016 provides additional useful design tips. For large gene insertion, homology arms with 300-1000 bp flanking the insertion gene have been reported. In most cases, 500 bp long homology arms should be sufficient. It is important to design your ssDNA template with a silent mutation to mutate the sgRNA PAM sequence in order to avoid secondary cleavage. As knock-in donor design can be complicated, we highly suggest that you use our HDR knock-in design tool to view and edit your sequence before ordering.

• What is the recommended homology arm length on each side of the template DNA when designing ssDNA?

  Based on internal testing, we recommend the following homology:

  • for point mutations and small insertions and deletions (a few nt): 40-50 nt HA
  • for longer insertions up to 100 nt: 70 nt HA
  • for insertion sizes larger than 100 nt: 300 nt HA

  It is important to note that knock-in efficiency may decrease as the insert size increases.

• What is the maximum ssDNA length that you can produce?

  We routinely produce sequences up to 3000 nt in length. We have also successfully synthesized ssDNA that are up to 5000 nt long with motifs of extremely high GC content and repeat regions. If you want to synthesize ssDNAs longer than 3000 nt long, please contact a CRISPR expert at crispr@genscript.com to get a quote.

• What is the maximum quantity that you can provide for ssDNA templates?

  We can deliver up to 2 mg per batch for GenExact ssDNA. However, the yield is dependent on the length and difficulty of the ssDNA sequence.

• What quality control tests do you provide for ssDNA templates?

  We carry out two primary QC tests on our RUO grade ssDNA products prior to lyophilization:

  • Sanger Sequencing: ensures the accuracy of the ssDNA 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 GenExact ssDNA templates.

• Why do you do double-sequencing, including the final ssDNA product?

  During the production of ssDNA, 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 ssDNA product. Then, we use direct sequencing on the final ssDNA product to confirm the ssDNA product homogeneity.

• How do you purify the final ssDNA product?

  We combine the advantages of magnetic bead and agarose gel purification to ensure high yield, absolute purity, and zero chemical contaminations.

• Do I need to worry about dsDNA contamination in the final ssDNA product?

  Absolutely not! Your final GenExact ssDNA product will only contain full length, sequence-verified, ssDNA molecules. By using our proprietary, patent-pending, enzymatic synthesis approach, we guarantee that our GenExact ssDNA products are delivered with non-detectable dsDNA levels.

• Why is there more than one band in the gel image on my COA report?

  Some ssDNA products may have strong aggregation tendency due to the nature of their nucleotide sequences. These aggregates are most likely formed due to intramolecular and/or intermolecular forces.

  To test whether the extra band in a gel image is ssDNA aggregates or dsDNA contamination, you can perform a digestion test using S1 Nuclease- which degrades ssDNA, but not dsDNA. If the ssDNA product is 100% pure, with no dsDNA, then all samples should be digested with S1 Nuclease and no band should be observed after running gel electrophoresis. However, if a product has dsDNA contamination, then it can't be fully digested after the addition of S1 Nuclease and a band will still remain present on the gel image.

  To further confirm whether the extra band is indeed ssDNA aggregates, you can cut out the ssDNA band on the gel and run a second around of gel electrophoresis. If the extra band is due to aggregated ssDNA molecules, it will still show up on the second gel after purification. In addition, the ratio of the extra band over the ssDNA band in the two gels would remain similar.
• What delivery formats are available?

  GenExact ssDNA 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 ssDNA?

  Our GenExact ssDNA are stable for at least 12 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 our GenExact User Instructions and our KI protocols.

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

  The quantity of ssDNA used for each test will depend on your specific application. For animal disease model generation purposes, 3 ug is usually sufficient. 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 ssDNA design?

  For ssDNA insertion, we recommend adding a 5’ Cas Targeting Sequence (CTS) and an annealing oligo for improved gene knock-in efficiency. Our CTS allows for the co-delivery of donor DNA and sgRNA/Cas9 RNP complex into the nucleus target gene site, therefore increasing the HDR knock-in rate.

  In our collaboration with Dr. Marson at Gladstone Institutes and UCSF, GenExact ssDNA paired with our CTS design (delivered via electroporation) consistently outperformed in-house generated HDR templates, showing lower levels of toxicity and a knock-in efficiency of 46.2% at a cGMP-compatible scale.

  Data from our collaboration with Dr. Brian Shy has shown that our unique CTS strategy, which involves adding the CTS to the 5' end of the ssDNA only, drives sufficient improvements in knock-in efficiency that are comparable to results from using both a 5' and 3' CTS.

  Learn more by reading the case study here.

• 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 ssDNA?

  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.