Diagram shows strategies of the PAINT 2.0 and PAINT 3.0 methods. 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. Both PAINT 2.0 and PAINT 3.0 utilize the PE2 (PE–Cas9/pegRNA) system for donor processing to generate MHFs. An additional programable nuclease, e.g., saCas9 is applied to introduce DSBs at the genomic target site. The left panel shows PAINT 2.0-mediated targeted KIs. A pair of 3′-MHFs flanking the transgene participates in targeted transgene integration without donor linearization, thus avoiding NHEJ-mediated imprecise KIs. The middle panel presents PAINT 3.0-mediated targeted KIs. The transgene cassette is sandwiched by a 3′-MHF on one side and a double-stranded HA on the other side. The 3′-MHF triggers strand invasion of the transgene while the HA mediates strand annealing to ligate the two genomic DNA parts. The right panel shows the generation of the 3′-MHF via a nicking & priming process. LMHF left micro-homologous flap, RMHF right micro-homologous flap.
