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When designing donor DNA for use in HDR, what are the optimal lengths of the left and right homology arms, and what is the maximum size of sequence that can be efficiently inserted in mammalian cells?
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, the length of the homology arms, the cell cycle, and the activity of the endogenous repair systems in the particular cell . These factors contribute to the high variability of HDR efficiency observed across different cell lines, and particularly in immortalized cells . Typically in replicating mammalian cells donor arms range are at least 500 bp in length . However, it is important to determine the optimal HDR conditions for your cell line.
Inserts between the homology arms are frequently in the 1–2 kb range . While longer inserts are possible, the efficiency of recombination decreases as the insert size increases . Finding successfully integrated inserts is likely to be challenging when inserts are greater than 3 kb in most mammalian cells.
Single-stranded oligo DNA (ssODN) has recently been identified as a substrate that is preferred by the HDR mechanism and often achieves good efficiency with homology arms as short as 40 bp [6, 7]. The drawback to using ssODNs is that they are limited in length to a few hundred bases, so the insert size is limited.
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