Figure 5.

A more comprehensive ExACT pathway. This redesigned ExACT pathway incorporates the two-step gap bridging mechanism and depicts the possibility for overlap between the multiple branching arms of the pathway. After CRISPR-Cas cleavage (1), the complex dissociates from the DNA. In the local absence of ssODN repair templates, the DNA ends are resected (A3). NHEJ and MMEJ occur to repair and relegate the broken DNA ends (A4.1 and A4.2), resulting in an error-prone final product containing indels. In the presence and accessibility of a repair ssODN, the oligo will either bind to both or just a single end of the broken DNA (B2 and C2, respectively). If both ends are stabilized by the ssODN, DNA synthesis and ligation to the opposite end are performed concurrently via DNA synthesis (B3). Once the oligo the dissociates (B4), the repaired strand can then be used as a template for a second synthesis step, repairing the other strand (B5). If only a single end associates with the ssODN, synthesis occurs from the 3′ end of the broken DNA, extending it using the ssODN as a template (C3). This extended end can then, once the oligo dissociates, bind to the opposite end of the cut site and bridge the DNA break. At this time or previously, the S strand of the downstream end is resected in a 5′ > 3′ direction (C4). Finally, the now-bridged DNA can be repaired via synthesis using the newly-synthesized upstream sequence as a template via a second round of synthesis. The final DNA break is then repaired wither via simple ligation or extension and ligation of the S strand sequence (C5).