Frequently asked questions

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Which repair pathway is most commonly used to repair CRISPR mediated double stranded breaks, NHEJ or HDR?

In most eukaryotic cells, the non-homologous end joining (NHEJ) pathway generates insertions and deletions during double-stranded break (DSB) repair. However, in the presence of a DNA template with homology to the sequences flanking the DSB location, homology-directed repair (HDR) can seal the DSB in an error-free manner[1]. In most cells, both of these repair pathways are active, however the HDR pathway is generally less efficient that the NHEJ pathway in the absence of a homologous template. This template is naturally present as sister chromatin in late S phase and G2 phase [2], and may be added artificially as donor DNA [3,4].
The efficiency of HDR is determined by the concentration of donor DNA present at the time of repair, the length of the homology arms of the donor DNA, the cell cycle, and the activity of the endogenous repair systems [4]. These factors contribute to the high variability of HDR efficiency observed across cell lines, particularly in immortalized cells [5]. It is important to determine the optimal HDR conditions for each cell line.
  1. Iliakis G, Wang H, et al. (2004) Mechanisms of DNA double strand break repair and chromosome aberration formation. Cytogenetic and Genome Research, 104(1–4):14–20.
  2. Heyer WD, Ehmsen KT, et al. (2010) Regulation of homologous recombination in eukaryotes. Annual Review of Genetics, 44:113–139.
  3. Elliott B, Richardson C, et al. (1998) Gene conversion tracts from double-strand break repair in mammalian cells. Molecular and Cellular Biology, 18(1):93–101.
  4. Lin S, Staahl BT, et al. (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 3:e04766.
  5. Hasty P, Rivera-Perez J, et al. (1991) The length of homology required for gene targeting in embryonic stem cells. Molecular and Cellular Biology. 11(11):5586–5591.