(A) Schematic illustration of the additional RGEN targeting sequences on the 2nd exon of DJ-1 gene. Target site no. 2 and 3 do not contain GG at their 5′ ends, while target site no. 1 starts with the sequence GG. The targeting sequence of sgRNA is indicated in green box, adjacent to NGG protospacer adjacent motif (PAM) sequence in light blue box. (B,C) The sequences of sgRNAs for target site no. 2 (B) and 3 (C). Red and green letters indicate the sequence required by T7 RNA polymerase and the customizable targeting sequence, respectively. Two sgRNAs were designed for each target site. The sgRNA no. 2a and 3a contain the 18-nts sequence complementary to their genomic target site, while the sgRNA no. 2b and 3b contain the 20-nts sequence. These sgRNAs also contain 1- or 2-nt mismatches to their genomic target sequence at 5′ end. (D) Heteroduplex mobility assay (HMA) in embryos injected with a mixture containing 100 ng/µL of Cas9 RNA and 25 ng/µL of sgRNA. Multiple heteroduplex bands were shown in PCR amplicons from each the RGEN-injected embryo, whereas a single band was shown from each “Control” embryo without the injection of the RGENs. (E–H) Subcloned sequences observed in the embryos injected with sgRNA no. 2a (E), 2b (F), 3a (G), or 3b (H). Red dashes and letters indicate the identified mutations. The sgRNA targeting sequence and PAM indicate in green and light blue boxes, respectively. The size of deletions and insertions are shown to the right of each mutated sequence (Δ; deletions, +; insertions). Numbers on the right edge indicate the numbers of mutated clones identified from all analyzed clones from each embryo.

(A) Schematic illustration of restriction fragment length polymorphism (RFLP) analysis to calculate mutation frequencies. The sgRNA no. 3a contatin an AluI restriction enzyme site (Red letters with underline) on the potential cleavage site indicated by an arrowhead. A 285 bp fragment amplified using primers DJ1-FW2 and DJ1-RV1 produces both 75 bp (a) and 210 bp fragments by AluI digestion in wild type. (B) Gel image of AluI-digested fragments analyzed in MultiNA system. The RGEN-injected embryos exhibited undigested fragments (a+b). Images from a representative embryo injected with varying amounts of Cas9 RNA and sgRNA no. 3a are shown. (C,D) Mutation rates at each injected Cas9 RNA concentration with 25 ng/µL of sgRNA (C) and at each injected sgRNA concentration with 100 ng/µL of Cas9 RNA (D). The mutation rate was calculated as the molar concentration of the undigested fragment (a+b) with AluI as a percentage of the sum of molar concentrations of the undigested fragment (a+b) and the larger digested fragment (b). The molar concentration of each fragment was quantified using the MultiNA Viewer software. Columns and error bars represent mean ± s.e.m. (n = 12). The different letters at the top of the columns indicate significant differences (P<0.05; one-way ANOVA followed by Tukey's HSD test).

(A,B) Heteroduplex mobility assay (HMA) of the mutagenized off-target loci OT2-I4 (OT2-II1) (A) and OT2-II4 (B). The upper panel shows the alignment of the off-target sites with the targeting sequence no. 2. The targeting sequence of sgRNA is indicated in green box, adjacent to NGG protospacer adjacent motif (PAM) sequence in light blue box. The lower panel shows HMA of the off-target loci using genomic DNA mixtures of the 12 embryos. Multiple heteroduplex bands were shown in PCR amplicons from embryos injected with 100 ng/µL of Cas9 RNA and 25 ng/µL of sgRNA no. 2a, whereas a single band was shown from “Control” embryos without injection of the RGENs. (C,D) Subcloned sequences of off-target alterations at OT2-I4 (OT2-II1) (C) and OT2-II4 (D) were identified in the RGEN-injected embryos. Red dashes and letters indicate the identified mutations. The sgRNA targeting sequence and PAM indicate in green and light blue boxes, respectively. The size of deletions and insertions are shown to the right of each mutated sequence (Δ; deletions, +; insertions). Numbers on the right edge indicate the numbers of mutated clones identified from all analyzed clones from each genomic DNA mixture.

Both the DJ-1 targeting locus and two disrupted off-target loci were analyzed using genomic DNA mixture of the 12 embryos that were injected with dilution series of sgRNA no. 2a and 100 ng/µL Cas9 RNA. (A) Heteroduplex mobility assay (HMA). Each analyzed locus and concentration of the injected sgRNA was shown in the upper side of the panel. (B,C) Subcloned sequence of the targeting locus observed in the embryos injected with either 10 ng/µL (B) or 5 ng/µL (C) of sgRNA no. 2a. (D–G) Subcloned sequence of the off-target locus OT2-I4 (OT2-II1) (D,E) or OT2-II4 (F,G) observed in the embryos injected with either 10 ng/µL (D,F) or 5 ng/µL (E,G) of sgRNA no. 2a. (B–G) Red dashes and letters indicate the identified mutations. The sgRNA targeting sequence and PAM indicate in green and light blue boxes, respectively. The size of deletions and insertions are shown to the right of each mutated sequence (Δ; deletions, +; insertions). Numbers on the right edge indicate the numbers of mutated clones identified from all analyzed clones from each genomic DNA mixture.

Each G0 founder that was injected with 100 ng/µL of Cas9 RNA and 25 ng/µL of sgRNA no. 1 was mated with wild-type fish to screen heritable mutations. Mutation sequences identified in each F1 embryo of them by heteroduplex mobility assay (HMA) and subsequent direct sequencing were shown. Red dashes and letters indicate the identified mutations. The sgRNA targeting sequence and protospacer adjacent motif (PAM) indicate in green and light blue boxes, respectively. The size of deletions and insertions are shown to the right of each mutated sequence (Δ; deletions, +; insertions). Numbers on the right edge indicate the numbers of mutated embryos identified from all analyzed embryos. The frequencies of mutations in each founder are indicated on the top of mutation sequences.