Figure 1: Generation of DJ-1/parkin/PINK1 triple gene-modified pigs using the CRISPR/Cas system.

(A) Schematic diagram of generation of triple gene targeted pigs by zygote injection of Cas9 mRNA/sgRNAs. In vitro transcribed Cas9 mRNA and multiplexing sgRNA were co-injected into the cytoplasm of one-cell stage pig embryos. Then, the injected embryos were transferred into recipient gilts to produce the genetically modified offspring. (B) Schematic diagram of sgRNAs targeting at DJ-1, parkin and PINK1 locus. The PAM sequences are highlighted in green. The sgRNA targeting sites are highlighted in red. (C) Sanger sequencing of the targeting site in mutant pigs. For each gene, the wild-type sequence is shown at the top with the target sites highlighted in red. At least 15 TA clones of the PCR products were analyzed by DNA sequencing. The change in length caused by each mutation is to the right of each sequence. The PAM sequences are highlighted in green; the mutations in blue; deletions (−), insertions (+).