Donor DNA Design and Confirmation
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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.
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.
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.
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.
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For research use only. Not intended for any clinical use.
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