Jose Arturo Gutierrez-Triana and colleagues from Heidelberg University report in eLIFE a CRISPR/Cas9 based methodology that simultaneously decreases donor DNA multimerization and increases HDR single-copy integration.
A common obstacle that CRISPR/Cas HDR mediated genome editing faces is the accurate and efficient insertion of a single copy sequence into a targeted genomic location. Often, the donor DNA that is injected is multimerized resulting in insertions of longer and/or non-functional DNA sequences. To overcome this obstacle, the authors designed modified shielded long dsDNA donors by blocking the 5’ ends of PCR amplified DNA using Biotin, Amino-dT (A-dT) or carbon spacers (SpC3) and including phosphothioate bonds between the 5′ terminal nucleotides.
These 5’ modified and unmodified DIG labelled long dsDNA donors were first tested by direct injection into the one cell stage of the Japanese rice fish, medaka (Oryzias latipes). As exogenous DNA is rapidly amplified during embryogenesis, the direct injection served as a test for multimerization. Total embryonic DNA was extracted 2, 4, and 6 hours post injection.
While multimers were detected as early as 2h in the unmodified and A-dT modified donors, no multimers were detected after 6 hours in the 5’ modified Biotin or SpC3 donors, suggesting that 5’ blocking can indeed decrease multimerization.
The authors then proceeded to test these donors in a targeted HDR CRISPR/Cas-9 experiment. GFP expressing cassettes were designed and injected (for three genes: a retinal homeobox transcription factor, rx2, a non-muscle cytoskeletal beta-actin (actb) and a DNA methyl transferase 1 (dnmt1). The frequency of HDR events was much higher in the embryos injected with the 5’ modified cassettes, with up to 9% of embryos displaying single copy HDR integration, which the authors were able to carry through to the F3 generation.
The methodology designed by Gutierrez-Triana et al. is a simple and elegant solution that improves CRISPR/Cas9 genome editing. Incorporating 5’ Biotin tags into the donor DNA was the best approach tested, reducing multimerization and increasing the chances of single copy insertion. This methodology works with donor DNA of different sizes and proved fragments of 2547bp can be inserted efficiently. Additionally, the authors report that 400 bp flanking the 5’ and 3’ regions should be sufficient for targeted HDR mediated CRISPR/Cas studies.
Gutierrez-Triana, J.A., Tavhelidse, T, Thumberger T., Thomas, I., Wittbrodt B., Kellner, T., Anlas, K., Tsingos, E., Wittbrodt, J. (2018). Efficient single-copy HDR by 5’ modified long dsDNA donors. Life 2018;7:e39468 doi: 10.7554/eLife.39468