DNA DSB repair. The central initiating event in gene editing is a DNA DSB, which may be resolved through 2 different branches of cellular DNA repair mechanisms. The first branch is known as classic NHEJ, which primarily acts to directly rejoin the broken ends. This may occur seamlessly, with the generation of small insertions or deletions at the ends of the break (red-colored DNA), or in the case that the gene-editing procedure involves provision of a piece of foreign DNA, by insertion of a “trans” template into the DSB (blue-colored DNA in the pictured “trans” insertion template represents nonnative DNA sequence, gray DNA represents regions of homology to each side of the DSB; these would be optional if the “trans template” were intended solely for targeted insertion, but would be included in a typical gene targeting construct). The second branch is known as HDR, which involves the resection at the 5′ ends of each side of the DSB, and use of the resulting single-stranded DNA ends in a homology search for complementary DNA sequences. This may result in identification of short strands of homology near the original break site, which, upon resealing of the ends, will typically result in small deletions through a process known as alternative NHEJ (note that this pathway may also lead to targeted insertion of foreign DNA). Alternatively, extensive resection may occur, following which more extensive regions of homologous base pairing are required to resolve the break through HDR with a trans or cis template. Professional illustration by Patrick Lane, ScEYEnce Studios.