Fig. 5

Generation of the pccb knockout mutants using CRISPR/Cas9 gene editing and behavioral assessment in zebrafishA Schematic representation of the zebrafish pccb gene structure. The protein comprises an MmdA domain of acetyl-CoA carboxylase and a crotonase-like domain of the crotonase/enoyl-Coenzyme A (CoA) hydratase superfamily. A one-nucleotide insertion allele was generated by injecting a single guide RNA (sgRNA) targeting exon 3 of the pccb gene. The asterisk (“*“) indicates the predicted premature stop codon resulting from the frameshift mutation, leading to early truncation of the protein before the functional domains. B Sequencing results comparing wild-type (pccb+/+) and mutants (pccbe3/+ and pccbe3/e3). C Predicted three-dimensional models of the pccb+/+ and pccbe3/e3 proteins generated using the SWISS-MODEL online tool. D Expression levels of pccb mRNA in brain tissues, indicating a significant reduction in the mutant zebrafish. E Schematic of the open field test setup and representative heatmaps illustrating movement patterns of zebrafish during the test. F Total distance moved during the open field test, showing increased locomotor activity in pccbe3/+ zebrafish compared to wild-type controls. G Average swimming velocity in the open field test (pccb+/+, n = 29; pccbe3/+, n = 36), demonstrating hyperactivity in the mutant fish. H Ratio of distance traveled in the center zone to the total distance moved, reflecting anxiety-like behavior. I Ratio of time spent in the center zone to the total time (pccb+/+, n = 20; pccbe3/+, n = 25), further indicating increased anxiety levels in pccbe3/+ zebrafish. Bars represent the mean ± SEM.