Fig. 2
The ins/del mutations. (a) and (b) Summary of the ins/del mutations detected in the LWSa (a) and LWSb (b) alleles. The target sequence of sgRNA-4 is highlighted in purple and blue. The inserted or deleted nucleotides are indicated by red characters or red hyphens, respectively. The total number of ins/del nucleotides and the number of F1 fish that inherited each mutation (of the 166 F1 fish analyzed) are shown on the right. (c) Summary of the double-ins/del alleles inherited to F1 fish. The allele names are based on the total number of ins/del nucleotides in each gene; e.g., “+2a + 5b” indicates two and five nucleotides were inserted in the LWSa and LWSb genes, respectively. Thus, three of these eight double-ins/del mutations (highlighted in red) cause double frameshifts on both the LWSa and LWSb genes. (d) RT-PCR images using the eyes. Because of the high sequence similarity between the LWSa and LWSb genes (see Fig. 1b), we did not discriminate between them for RT-PCR analysis, and an identical pair of primers was used for simultaneous amplification of the LWSa and LWSb transcripts. The primers sandwich an intron (87 bp) and, therefore, bands at 375 bp and 462 bp are products from cDNA and genomic DNA, respectively. Considering that the 462-bp products are preferentially amplified in lws +2a+5b (while they are not amplified at all in control), transcription of LWSa/b in the lws +2a+5b mutants seems to be very weak in comparison with that in the control fish. A larger band (>500 bp) found in all lanes could be non-specific