ssDNAs donor templates demonstrated significantly improved editing efficiency and specificity, as well as reduced off-target integration, when compared to double-stranded DNA donors, especially in editing primary cells, stem cells, and developing transgenic animal model.1-4.

Recent studies demonstrate that single-stranded DNA (ssDNA or ssODN) is the best donor template for CRISPR homology directed repair (HDR) template for creating gene knock-in, with high editing efficiency and reduced off-target events.

1. “While the ssDNA repair template requires shorter homology arms and is inserted at a very high efficiency, the dsDNA repair template requires long homology arms and is inserted with much lower efficiency.” Miura H., et al., Sci. Rep 2015 Aug 5;5:12799. doi: 10.1038/srep12799.

2. “[...] long ssDNA donors (instead of dsDNAs) are central to obtaining consistently higher success in CRISPR animal genome engineering.” Quadros R.M., et al., Genome Biol. 2017 May 17;18(1):92. doi: 10.1186/s13059-017-1220-4

3. “Similar to what has been described with viral HDR templates, we found evidence to suggest that double-stranded templates could integrate independent of target homology, albeit at low rates. These rare events could be reduced almost completely by using single-stranded DNA (ssDNA) templates." Roth T.L., et al. Nature 2018 Jul;559(7714):405-409. doi: 10.1038/s41586-018-0326-5.

4. “ssCTS templates increased knock-in efficiency, live cell counts and absolute yield of knock-in cells across all primary human cell types evaluated here including CD4+ T cells, CD8+ T cells, regulatory T cells (Treg), NK cells, B cells, CD34+ HSCs and gamma-delta T cells (γδ).” Shy B.R, et al., Nat Biotechnol. 2022 Aug 25. doi: 10.1038/s41587-022-01418-8.