Fig. 4.

ZFN-driven repair of endogenous DSBs and of ZFN-induced breaks followed by homology-directed targeted integration. (A) SDSA-based HDR of an endogenous break (32). After a DSB a single-stranded chromosomal tail invades a sister chromatid, and after DNA synthesis of a short stretch the D loop collapses the newly synthesized DNA. (B) Homology-directed targeted integration after a ZFN-induced break and SDSA-based transfer of genetic information into the break, a model most consistent with the data presented in the current work and evidence in the literature. After a ZFN-induced break, the single-stranded chromosome end homes into the homology arm carried by the donor (this process is unimpeded by the presence of the insert because the latter is located precisely at the position corresponding to the break and hence remains “invisible” to the homology search mechanism). Synthesis then proceeds across at least 50% of the insert length, with the newly synthesized single-stranded DNA trailing the D loop. Irrespective of whether the two broken chromosomal ends use the same (as shown) or two different donor molecules as templates, once synthesis in each D loop has proceeded long enough for the two DNA stretches to overlap, the newly synthesized DNA molecules can leave the D loop, anneal to each other, and then use each other as templates to restore an intact chromatid, now carrying the donor-specified transgene at the chromosome.