Investigating and comparing the sustainability practices and awareness of consumers in selected Melbourne suburbs

Mozambique tilapia (Oreochromis mossambicus), an invasive freshwater species in Australia since the 1970s, poses a substantial threat to local ecosystems and economies. Traditional physical and chemical population control methods have proven to be ineffective, driving the need for more sustainable biological control methods including viral and genetic biocontrol strategies. Building on prior research on a novel genetic biocontrol technology called Self-Stocking Incompatibility System (SSIMS) using CRISPR-Cas9 targeting wnt1 gene in zebrafish, this study aims to identify and characterize wnt1 gene in O. mossambicus. The study involved computational analysis, experimental validation of wnt1 using PCR and DNA sequencing, and an assessment of guide RNAs efficiency using an in vitro CRISPR-Cas9 cleavage assay. In silico analyses successfully identified wnt1 gene region “Oreochromis_mossambicus_EIV1_scaffold_0000010” spanning 6137920-6143233 within the O. mossambicus genome assembly and revealed a gene structure comprising four exons in the negative strand of the genome. The identification of the promoter region using the Berkeley Drosophila Genome Project (BDGP) and the presence of common ancestral transcription elements emphasize its regulatory significance. PCR amplification of the gene sequence in cell cultures and muscle of O. mossambicus yielded nucleotide sequences of 4159 bp and 4140 bp, respectively. This gene was predicted to encode 370 amino acid products and pairwise sequence alignment showing 99% similarity with those of O. niloticus, a closely related species within the tilapia family (Cichlidae), and zebrafish which belong to a different family (Cyprinidae), suggesting high conservation of wnt1 gene across fish species. In vitro cleavage assay with CRISPR-Cas9 showed the efficiency of guide RNAs targeting exon 2 and exon 3 of wnt1 gene, cutting the gene at the expected locations. This study lays the foundation for potential gene knockouts in vivo in O. mossambicus, enabling future progress towards translation of SSIMS technology from zebrafish model