Donor DNA Design and Confirmation
Cas9-sgRNA complex gene editing is an effective technique for obtaining insertion, removal and mutation with high efficiency. But applying this technology to engineer site-specific insertion and tagged of genes or sequences longer than 200 bp via homology-directed repair (HDR) is often very difficult. Specifically, employing readily synthesized donor DNA oligonucleotides can be efficiently generated and transmitted through the Drosophila germline within short time making the promise of larger tag or reporter, etc.
CD BioSciences provides a series of donor DNA oligonucleotides service, both single stranded DNA (ssDNA) and large double stranded DNA (dsDNA). Our state-of-the-art services improve and extend your study needs including target sites selection, sequence design, synthesis, validation of efficiency and off-target analysis relying on the characteristics of Drosophila CRISPR/Cas9 system. We are always competent to offer tailor-made approached to accomplish every request to oligo synthesis.
What does donor DNA do in CRISPR?
Donor DNA plays the same role as homologous DNA employed in homology-directed repair (HDR) for precise repair. In Drosophila, the donor DNA can take two forms: (1) ssDNA oligonucleotides synthesized up to 2 kb in length and used to integrate short sequences, or (2) dsDNA constructs containing hundreds to thousands of nucleotides of homologous sequence on either side of the double-strand breaks (DSB) site.
Using DNA donor templates indicates obvious advantage that they can be synthesized rapidly in vitro, obviating additional budget. Besides, ssDNA significantly reduces the possibility of random and off-target integration into the genome, thereby increasing the efficiency of gene editing. However, the size of ssDNA is generally limited to 2 kb. dsDNA donor templates can be employed in targeting vector to efficiently incorporate larger exogenous sequences (~5 kb) or modify large region sequences at higher efficiency than short oligonucleotides.
Our broad portfolio is backed by a range of cutting-edge technologies and scientists' counsel, providing and supporting clients with appropriate Drosophila donor DNA solution. In Drosophila donor DNA design strategy, we usually add 60 nt homology arms to ssDNA and 1 ~1.5 kb homology arms to dsDNA as plasmid. The homology arm of dsDNA is amplified from the cas9 transgenic line which will be targeted. All the donor DNAs should no longer be recognized by their respective sgRNAs once they are integrated, which is meant that introducing mutations in the PAM or 3' guide sequence is not appropriate.
Fig. 1 ssDNA design for CRISPR/Cas9 gene editing (Gratz SJ et al. 2015) Donor DNA Design and Confirmation Service
To facilitate high-quality gRNA design, we offer a professional general CRISPR donor DNA strategy includes mutagenesis, insertion, deletion, incorporation, and desired modification to serve you well-workable designed products.
| Service | Description |
|---|---|
| Donor DNA design and synthesis | Design Donor DNA based on clients' destination, targets sites 5' and 3' homology arms and genes sequence analysis (at least 3 Donor DNA) |
| Donor DNA construction | 1. Donor DNA synthesis (clone or PCR) 2. Appropriate digestion (for ssDNA) |
| Vector construction (Optional) | Choose the optimal vectors according to your research requirements. |
Why CD BioSciences?
- We have record-proven expertise and experience in donor DNA design, generation, and optimal insertion site based on Drosophila genome and the characteristics of target genes.
- We build comprehensive targeting donor DNA library amplified from Cas9 transgenic Drosophila lines, which not only includes reporters (e.g., GFP, RFP, mCherry), markers (e.g., mini-white, vermillion, yellow), specific-site sequences and recombinase (e.g., Flp-FRT, phiC31-att). We provide customized services for the genes you want to insert.
- Through our mature and professional platform, high-quality donor DNA with detailed evaluation report will ship to you in fast turnaround time.
- We are proud of our best after-sale service, allowing further CRISPR/Cas9 custom strategy for every gene in Drosophila, such as injection concentration, method, off-target analysis and donor DNA optimization
CD BioSciences can design guides not just for protein or RNA tagging but also for mutant libraries, and transcriptional reporters. If you could not find what you really want in the website, please feel free to contact us.
References
- Gratz SJ, et al. (2015). CRISPR-Cas9 Genome Editing in Drosophila. Curr Protoc Mol Biol. 111: 31.2.1-31.2.20.
- Gratz SJ, et al. (2015). Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9. Methods Mol Biol. 1311:335-348.
For research use only. Not intended for any clinical use.
Related Services