Engineered RGNs induces heritable gene disruption.

<p>(A) Schematic illustration of the RGN system. Engineered sgRNA:Cas9 system is depicted here based on the target sequence of the <i>fh</i> gene. sgRNA interacts with the complementary strand of the DNA target site harboring a 3′ protospacer adjacent motif (PAM) sequence (NGG) (yellow and red underlined text, respectively). sgRNA also interacts with Cas9 endonuclease (blue shape), resulting in DNA double-strand breaks (DSBs) at the target site. The reverse complement of the target site is highlighted as green text and the reverse complement of the PAM site is shown as red underlined text. The potential cleavage sites of Cas9 are indicated by arrowheads. This graphic representation is modified from a previous publication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone.0068708-Hwang1" target="_blank">[11]</a>. (B) Mutation frequencies in the germline induced by engineered RGNs. Fish that have been injected with sgRNA and Cas9 mRNA at the 1-cell stage were screened for founders. The concentrations of the sgRNA and the Cas9 mRNA are as indicated. The somatic mutation rates induced by these combinations have been reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone.0068708-Hwang1" target="_blank">[11]</a>. The percentages of the injected embryos that developed normally at 1 day post-fertilization are shown. The sequences of the indel mutations identified in the germline are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone-0068708-g002" target="_blank">Figure 2</a>.