Picking the right DNA donor template for CRISPR
Picking the right donor template can significantly affect the outcome of your CRISPR experiments or therapies. There are three main choices, plasmid or double stranded DNA (dsDNA), adeno-associated virus (AAV) or single stranded DNA (ssDNA). In this article, we will briefly go through each one as well as explaining how Moligo has changed the way ssDNA can be used.
Plasmid or double stranded DNA (dsDNA)
Plasmid DNA is most easily produced in the lab. It is simple to clone the sequence for your donor template into a plasmid and transfect this into cells. While they are the easiest to produce, plasmids contain a lot of extra DNA which can be problematic as in some instances, plasmid DNA can become incorporated into the host cell DNA at the CRISPR cut sites or even at off-target sites. One solution to overcoming this is to use linear, single stranded DNA that is produced as a PCR product although these are less stable. Both linear and plasmid DNA have relatively low efficiency compared to other types of donor template. However, the ease with which they can be produced means that they are often perfect for initial pilots and early screening. Until recently, dsDNA was also the most convenient donor template if longer CRISPR edits were needed. It is essential, even for early screens that highly pure DNA is used to avoid any toxicity or adverse effects on your cell lines.
Adeno-associated virus (AAV)
AAVs are extremely useful DNA delivery vectors. They are non-pathogenic viruses with a single stranded DNA genome. Recombinant AAVs can be made with all of the viral genes removed, and the only DNA packaged in the capsid is the sequence for the donor template. AAVs have been shown to have extremely high knock-in efficiencies as the virus has a natural ability to stimulate homologous recombination.
Another useful feature of AAVs is the protection and specificity they give to your donor DNA template. While encapsulated in the viral capsid, the donor DNA template is protected from degradation until the moment that it is released inside the cell. Furthermore, different AAV serotypes have different tissue tropisms. This means that it is possible to selectively target specific cell types or tissues -- as long as there is a recombinant viral serotype for that particular tissue.
The limitations for AAVs are that they are relatively expensive and time consuming to produce compared to dsDNA and ssDNA. The length of the donor template is also restricted by the packaging capacity of the virus which is less than 5 kb.
Single stranded DNA (ssDNA)
Using ssDNA has a reputation as being the go-to donor template for editing single bases or smaller regions of the genome. This is because while the length of ssDNA oligonucleotides (ssODNs) has been limited until recently, ssDNA has several important advantages over dsDNA for use as a CRISPR donor template. Firstly, the editing efficiency of CRISPR with ssODNs is improved meaning that less DNA material is needed which reduces costs as well as potential cytotoxic effects. The specificity of ssODNs is also improved as off-target effects are observed far less in studies with ssDNA compared with dsDNA.
Which donor template should you choose?
AAVs have some specific and important advantages such as their ability to package and securely deliver upto 5Kb of ssDNA. However their complexity coupled with a time consuming and costly production method means they are often not an optimal donor to use at the beginning of a study. It would be more efficient to start with either a dsDNA or ssDNA donor template and move to AAVs once you knew that you needed the cell tropism properties of a specific serotype or the increased knock-in efficiencies that may be gained by using a viral vector.
Then the question becomes, should you use dsDNA or ssDNA?
Conventionally, this was a question of the length of the donor template that was needed. Templates longer than 200 bases would require dsDNA as it wasn't possible to produce viable ssDNA templates. If only small edits were required, then ssDNA was used as it had higher efficiencies with less negative consequences like toxicity and off-target effects.
However, at Moligo Technologies, we are happy to be able to challenge this conventional approach. Using our patented method, we are able to produce ssDNA of up to 10 Kb. These are the longest of any DNA oligos on the market meaning that ssDNA now has a viable use in much larger CRISPR edits than were previously thought possible. By increasing the length of the DNA, it is possible to edit entire genes while having enough flexibility to include long homology arms for efficient and accurate recombination.
The single stranded DNA that we produce has many more benefits that make it an exciting option as a donor template for many new CRISPR applications. We ensure that our ssODNs are 99.999% pure to reduce the risk of any cell toxicity. We can also produce the ssODNs at industrial scale meaning that you have flexibility in what concentration of DNA you use and how many cells you can treat at once. While our ssONDs are long, they can be ordered with any sequence complexity so that the DNA you order from us will reflect the complexity of real genes. Finally, our ssODNs can be functionalised at a high density which can be very useful for reporting and validation as well as targeting.
If you would like to find out more about how we can provide you with long and pure ssDNA please contact one of our experts.
Talk to one of our experts
References
Russell DW & Hirata RK (1998). Human gene targeting by viral vectors. Nat Genet 18:325-330.
Gaj T, et al. (2016) Genome engineering using adeno-associated virus: basic and clinical research applications. Mol. Ther 24:458–464.
Gaj T, et al. (2017). Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.
Nucleic Acids Research 45(11) e98 DOI: 10.1093/nar/gkx154
Dever DP, et al. (2016). CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 539:384–389.