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Fig. 4 PEX1 mutant is generated by using CRISPR/Cas9 system. (A, B) The efficiency of guide RNA (sgRNA) was analyzed by T7E1 assay. Target sequence of sgRNA for genomic editing was designed based on the exon which has start codon of PEX1 (A). The mixture of synthetic mRNA of Cas9 and sgRNA for PEX1 was microinjected into fertilized eggs of zebrafish. Genomic editing identified in Cas9/sgRNA-injected embryos by T7E1 endonuclease at 24-hpf (B). Uncut represents PCR products without T7E1 digestion. (C, D) Genomic editing was identified in the F2 generation. Sequence analysis confirmed a founder zebrafish which has 2-bp deletion in target sequences (C). The amino acid sequence predicted from the mutated PEX1 sequence and that of wild-type (C). Arrowhead indicate NdeI cleavage site (C). RFLP assay showing the PCR amplicons of PEX1+/+, PEX1+/− and PEX1−/− digested with NdeI restriction enzyme in 24-hpf-old zebrafish embryos (D). M, 100-bp DNA size marker. RFLP, Restriction fragment length polymorphism.