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Frequently asked questions

Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Filter using one or more categories to focus on specific topics, or use the search bar to perform a text search.

When designing donor DNA for use in HDR, what are the optimal lengths of the left and right homology arms?

DNA with homology to the sequences flanking a double-stranded break (DSB) can serve as template for error-free homology directed-repair (HDR) of the DSB. The efficiency of HDR is determined by the concentration of donor DNA present at the time of repair, length of the homology arms, cell cycle, and activity of the endogenous repair systems in the particular cell [1]. These factors contribute to the high variability of HDR efficiency observed across different cell lines, particularly in immortalized cells [2].

The length of the inserted sequence (between the homology arms) is frequently in the 1–2 kb range [3], or shorter. While longer inserts are possible, the efficiency of recombination decreases as the insert size increases [4]. Finding successfully integrated inserts is likely to be challenging when inserts are greater than 3 kb in most mammalian cells. Inserts in the 1–2 kb range can be introduced by using double-stranded DNA (dsDNA) Alt-R™ HDR Donor Blocks from IDT. These are available in lengths ranging from 200–3000 bp and contain chemical modifications within universal, non-integrating terminal sequences to help reduce unwanted non-homologous (blunt) integration events. We recommend using 200-300 bp homology arms for our Alt-R HDR Donor Blocks.

Single-stranded oligonucleotides (ssODN) have recently been identified as a substrate that is preferred by the HDR mechanism and often achieves good experimental efficiency with homology arms as short as 40 bp [5,6]. But ssODNs are limited in length to a few hundred bases which limits the insert size. When using Alt-R™ HDR Donor Oligos as templates for a short insertion, tag, or SNP conversion, we have found arm lengths of 30–60 nt to be sufficient.

References

[1] Elliott B, Richardson C, et al. (1998) Gene conversion tracts from double-strand break repair in mammalian cells. Mol Cell Biol, 18(1):93–101.

[2] Lin S, Staahl BT, et al. (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 3:e04766.

[3] Dickinson DJ, Ward JD, et al. (2013) Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods,10(10):1028–1034.

[4] Li K, Wang G, et al. (2014) Optimization of genome engineering approaches with the CRISPR/Cas9 system. PloS One, 9(8):e105779.

[5] Chen F, Pruett-Miller SM, et al. (2011) High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods, 8(9):753–755.

[6] Davis L and Maizels N (2014) Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair. Proc Natl Acad Sci U S A, 111(10):E924–932.