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When designing donor DNA for use in homology directed-repair (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, 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, and particularly in immortalized cells [2]. Typically, in replicating mammalian cells, donor arms are at least 500 bp in length [3]. 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 [4]. While longer inserts are possible, the efficiency of recombination decreases as the insert size increases [5]. 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. When using Ultramer® 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. Thomas KR, Folger KR, Capecchi MR (1986) High frequency targeting of genes to specific sites in the mammalian genome. Cell, 44(3):419–428.
  4. Dickinson DJ, Ward JD, et al. (2013) Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods,10(10):1028–1034.
  5. Li K, Wang G, et al. (2014) Optimization of genome engineering approaches with the CRISPR/Cas9 system. PloS One, 9(8):e105779.
  6. 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.
  7. 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.
Tags:
  • Cas9
  • Alt-R
  • CRISPR
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