Figure 2

Generation of the tnfaip1 mutant in zebrafish using the CRISPR/Cas9 system. (A) Gene structure of tnfaip1 and location of CRISPR/Cas9 targets (red arrow), Target 1 and Target 2 are two knockout target sgRNAs, with the specific target sequence shown in black and the PAM (NGG) sequence in red. (B) The DNA sequencing peak and sequence of the tnfaip1 mutant. DNA sequencing of the corresponding genomic region of zebrafish tnfaip1 revealed a 160 bp deletion mutation. (C) The tnfaip1⁻¹⁶⁰ mutation resulted in a gene frame shift and early termination of the encoded protein. This mutation resulted in a protein truncated to 30 aa. The asterisks mark the termination codon. aa: amino acids. (D) Identification of genotype by DNA electrophoresis. +/+, wild type; +/−, heterozygote; −/−, homozygote. (E) Relative expression of tnfaip1 mRNA at 72 hpf detected by qPCR and 18SrRNA as an internal control. “*” represents p < 0.05, analyzed with unpaired two-tailed t-tests. (F) In situ hybridization to detect expression of tnfaip1 in the mutants. (G) Decreased protein levels in tnfaip1^−/− embryos at 72 hpf as measured by Western blot. Tubulin as an internal control.