A Rad51-independent pathway promotes single-strand template repair in gene editing
Fig 7
Model of heteroduplex formation and incorporation of mismatches during SSTR.
Fate of an ssODN with 4 mismatches either 5’ to the XhoI site (A) or 3’ (B). After the DSB is created and resected only the strand on the right can anneal to the template. This annealing creates a heteroduplex that may be repaired by the mismatch repair machinery including Msh2. Heterologies close to the 3’ end of the invading strand, but also at the 3’ end of the ssODN, can be excised by the 3’ to 5’ exonuclease activity of DNA polymerase δ. Only if the heteroduplex is converted to the template strand genotype will these mismatches be incorporated into the SSTR product (B). Mismatches 5’ to the XhoI site will be obligately copied by DNA polymerase after strand invasion (A). The dissociation of the newly copied strand allows it to anneal with the resected second end of the DSB, creating an obligate heteroduplex. Dissociation of the newly copied strand may occur without copying the end of the ssODN template. Heteroduplex DNA may then be corrected to the genotype of the donor template or left as unrepaired heteroduplex. In the absence of Msh2, most outcomes will have heteroduplex to the left of the XhoI site but no incorporation to the right, resulting in sectored colonies.