Targeted genomic modifications using genome editing technologies. DNA double-strand breaks (DSBs) induced by genome editing technologies are repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and homologous recombination (HR). NHEJ repair, which connects the ends of the broken strands, leads to unpredictable insertion and/or deletion mutations (green bar), while MMEJ repair uses microhomology sequences (yellow box) and often causes a predictable small deletion. HR repair requires long double-strand DNA fragments (blue bar) that possess homology to the targeted genomic locus. Site-specific integrations of donor DNA are mediated by these DNA repair mechanisms.

Strategy for establishing knock-in fish. Donor vector, gRNAs and Cas9 mRNA are injected into zebrafish embryos. The knock-in event is estimated by examining the expression of the fluorescent gene (green area). Potential F0 founders are mated with wild-type (WT) fish, and the knock-in lines expressing the fluorescent gene are selected. The targeted knock-in at the targeted locus is determined by genomic PCR and sequencing analysis.

Site-specific insertion of the hsp promoter-Gal4 donor into the evx2 locus. The donor vector consists of a bait sequence (blue box; target site B for Gbait-gRNA), the hsp promoter (light blue box), a Gal4 driver (pink box) and a polyA signal (pA) (orange box). The target site A for evx2-gRNA (red box) is located in the promoter region of the evx2 gene. When the CRISPR/Cas9 system simultaneously cleaves the target site A and B, the Gal4 driver is unpredictably integrated into the evx2 locus. Not only forward and reverse integrations but also tandem donor vector integrations can occur. We observed eGFP expression in a subset of neurons of the Tg[evx2-hs:Gal4]; Tg[UAS:eGFP] transgenic line because Gal4 driven by the endogenous evx2 enhancer induced eGFP expression as described previously [41]. Gray boxes; exons in the evx2 gene.

Precise site-specific integration of eGFP into the krtt1c19e locus. The donor vector consists of two bait sequences (blue box; target site B for Gbait-gRNA), a left homology arm (yellow box; 40 bp), and an eGFP (green box) and polyA signal (pA) (orange box) and right homology arm (light green box). The target site A for krtt1c19e-gRNA (red box) is located near the stop codon of the krtt1c19e gene. When the CRISPR/Cas9 system simultaneously cleaves the target sites A and B, the eGFP reporter between the microhomology arms is precisely integrated into the krtt1c19e locus. Broad eGFP expression was detected in the epidermis of the injected F0 embryo as described previously [44]. Gray boxes; exons in the krtt1c19e gene.