Figure 5
Efficient disruption of tilapia aldh1a2and cyp26a1by CRISPR/Cas9.
A-B, disruption of aldh1a2 (A) and cyp26a1 (B) were performed by CRISPR/Cas9. Gene structure, the target sites and the restriction enzyme cutting site (underlined) are shown. At 72 hours after injection, 20 embryos were randomly selected and pooled to extract their genomic DNA for PCR amplification and the mutations were confirmed with two assays, restriction enzyme digestion (Acc I and Hpy188 I) and Sanger sequencing. The Cas9 and gRNA were added as indicated. For both genes, an intact DNA fragment (indicated by the white arrow) was observed in embryos injected with both Cas9 mRNA and target gRNA. The percentage of uncleaved bands was measured by quantifying the band intensity. The Sanger sequencing results from the uncleaved bands are listed. Deletions are indicated by dashes. The proto-spacer adjacent motif (PAM) is highlighted in green. Numbers to the right of the sequences indicate the loss or gain of bases for each allele, with the number of bases deleted (−) indicated in parentheses. WT, wild type. C-D, the expression of aldh1a2 in control and Aldh1a2-deficient fish (C), the expression of cyp26a1 in control and Cyp26a1-deficient fish (D) as measured by real-time PCR. Less amplification of aldh1a2 and cyp26a1 were detected compared to the control gonads. Data were expressed as mean ± SD (n = 5). * represents a significant difference at P < 0.05 for comparisons between control and deficient fish that were investigated by one-way ANOVA with a post-hoc test.
