NHEJ vs HDR: Which repair pathway to repair CRISPR mediated double stranded breaks?
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.
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 , 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 . These factors contribute to the high variability of HDR efficiency observed across cell lines, particularly in immortalize cells . It is important to determine the optimal HDR conditions for each cell line.
- 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.
- Heyer WD, Ehmsen KT, et al. (2010) Regulation of homologous recombination in eukaryotes. Annual Review of Genetics, 44:113–139.
- 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.
- Lin S, Staahl BT, et al. (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 3:e04766.
- 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.