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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. 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 immortalized cells . It is important to determine the optimal HDR conditions for each cell line.
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