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Single-stranded DNA (ssDNA or ssODN) has been proven to be the most effective CRISPR homology-directed repair (HDR) template for creating gene knock-ins, offering high editing efficiency and reduced off-target integration. GenScript provides high-quality, sequence-verified ssDNA to maximize the editing efficiency of your CRISPR experiments.
Dr. Theodore Roth
the Marson Lab at University of California San Francisco
Long ssDNA sequences are difficult to produce in the lab, especially at the high concentrations necessary for gene editing experiments. We were able to successfully integrate large DNA sequences into primary human T cells using GenScript's long ssDNA product.
|
Quantity (µg) |
GenExact™ ssDNA Length (nt) | |||||
|---|---|---|---|---|---|---|
| ≤500 | 1000 | 2000 | 3000 | 4000 | 5000 | |
| 3 | $400 | $675 | $1,300 | $1,950 | $2,450 | $2,950 |
| 5 | $500 | $800 | $1,500 | $2,250 | $2,850 | $3,450 |
| 10 | $650 | $975 | $1,700 | $2,500 | $3,200 | $3,900 |
| 20 | $750 | $1,100 | $1,900 | $2,800 | $3,600 | $4,400 |
| 50 | $1,200 | $1,600 | $2,650 | $3,950 | $5,050 | $6,150 |
| 100 | $1,500 | $1,925 | $3,100 | $4,600 | $6,100 | $7,600 |
Delivered in as fast as 3 weeks.
* We also offer <200nt ssDNA through our DNA Oligo Chemical Synthesis Service.
| Test Specification | Detection Method | Release Criteria | Research Grade (≤2mg) |
|---|---|---|---|
| Purity | Agarose gel electrophoresis | Single band |
✔
|
| Sequence accuracy | Sanger sequencing | 100% sequence alignment |
✔
|
| Optical density | Spectrophotometer at 260 nm/230 nm | ≥ 2.0 |
✔
|
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
All-encompassing non-viral solutions
RUO to cGMP linear ssDNA, dsDNA and miniaturized circular dsDNA
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
Collaboration with Marson's lab from UCSF (University of California San Francisco) showed that:
Brian R Shy. et.al., bioRxiv(2021), Read the whole article.
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
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.
Based on internal testing, we recommend the following homology:
It is important to note that knock-in efficiency may decrease as the insert size increases.
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.
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.
We carry out two primary QC tests on our RUO grade ssDNA products prior to lyophilization:
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.
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.
We combine the advantages of magnetic bead and agarose gel purification to ensure high yield, absolute purity, and zero chemical contaminations.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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