Figure 1

Gene targeting strategies using targeted double-strand breaks. When chromosomal DNA is cleaved (red arrowhead), the resulting double-strand break is repaired by non-homologous end joining (NHEJ) or by homology-dependent repair (HDR). NHEJ may result in perfect rejoining, of the ends, or in the introduction of point mutations and indels (knock-out). NHEJ may also join exogenous linear DNA (shown in yellow) to the broken ends of the chromosome (homology-independent knock-in); the orientation and reading frame in these insertions is random, unless directed by complementary overhangs [42, 44, 45]. HDR repairs the double-strand break by precise copying of a repair template carrying an exogenous sequence (shown in yellow) flanked by sequences with homology to the targeted locus (in blue) (homology-dependent knock-in). The repair template usually consists of circular plasmid DNA with long homology arms [46–50] or short single-stranded oligonucleotides (ssODNs) bearing 10 to 40 nucleotides of homologous sequence at each end [48, 50–52].