Diagram shows HDR-, NHEJ-, and PAINT-mediated targeted integration of foreign DNAs. 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. HDR-based integration (first from the left) requires long homologous arms typically ranging from 500-2000 bp, and is facilitated by the introduction of a DSB at the genomic target site. The NHEJ-base method (second from the left) requires concurrent excision of the transgene cassette and cleavage of the genomic DNA at the target site. The linearized double-stranded transgene then integrates into the genomic target site bidirectionally via the NHEJ repair pathway. For the PAINT method (second from the right), both 3'-ends of the excised transgene are modified with single-stranded micro-homologous overhangs (MHOs) by the spCas9-RT/pegRNA system, which then dictate efficient and unidirectional integration of the transgene. The last panel is a diagram showing the synthesis of the right 3'-MHO at the end of the excised transgene. LHA left homologous arm, RHA right homologous arm, LMHO left micro-homologous overhang, RMHO right micro-homologous overhang, RT reverse transcriptase, PBS primer binding site.
