Diagram compares the working models of plasmid donor-templated HDR and PAINT. Each line corresponds to a DNA strand. LHA and LMH are marked in red; RHA and RMH are marked in cyan. Triangles mark the specific cleavage sites in the genome and donors. Comb teethes represent the annealing of homologous strands. The left panel shows the SDSA model of plasmid donor-templated HDR. There are three central steps of the SDSA pathway. End resection, the 3′-ended DNA strand is resected at the break to create a 3′ single-stranded HA. Strand invasion, the single-stranded HA invades the plasmid donor by displacing one strand of the homologous DNA duplex and pairing with the other. This step then initiates donor-templated DNA repair synthesis. Annealing, the synthesized HA homologue then repairs with the homologous genomic DNA end to mediate DNA end rejoining. For plasmid donor-templated HDR, extended DNA end resection is a crucial step but is rate-limiting. The right panel presents a working model of PAINT method. PAINT achieves high-efficiency targeted transgene integrations mainly through PMEJ, an SDSA mechanism similar to HDR. In this PMEJ model, it is the single-stranded MHOs of the primed donor that invades the homologous genomic DNA ends to initiate DNA repair synthesis. This process is independent of extended genomic DNA end resection and, thus, may contribute to elevated KI efficiencies. A small portion of NHEJ-based transgene incorporation may also be involved in PAINT-mediated targeted KI. LHA left homologous arm, RHA right homologous arm, LMHO left micro-homologous overhang, RMHO right micro-homologous overhang. For b , e , editing efficiencies were measured as the percentage of EGFP + cells among total transfected cells. Three replicates were performed. The results are presented as the mean ± SEM. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’ s t -test, two-sided.
