Targeted insertion of mloxP mediated by Cas9/gRNA in zebrafish. (A) Cartoon showing the principle of inserting mloxP into a target site in the etsrp locus. The primers for primary PCR genotyping of zebrafish embryos with the expected mloxP insertion being marked. Gel image showing 4 out of 12 randomly selected Cas9/gRNA-treated embryos at 50 hpf that had the expected mloxP insertion (arrows). (B) Sanger sequencing result of the PCR amplicon from one representative Cas9/gRNA-injected zebrafish embryo at 50 hpf, with the correct insertion of mloxP in the etsrp locus as domonstrated in A. PCR fragments were amplified from the tested embryo and cloned into the pEASY vector for sequencing (20-30 clones per embryo), and one clone was confirmed to have the correct insertion of mloxP in the etsrp locus. 1, 3, 4 or 6 represents the original sequence in the left or right arm, whereas 2 or 5 highlights unexpected sequences (red).