There exists a need to construct intraspecific genetic barriers to reproduction in a variety of important fish species. In food-fish aquaculture, this would mitigate the risk of undesirable breeding between farmed escapees and their wild conspecifics. In invasive fish management , these genetic barriers can be erected to create reproductively isolated strains for genetic biocontrol. Here we present engineered genetic incompatibility (EGI), a genetic strategy to produce intraspecific incompatibility . EGI relies on CRISPR activation (CRISPRa) to induce lethal gene expression in the hybrid offspring of a wild type/ EGI cross. Our lab has demonstrated EGI’s effectiveness in D. melanogaster and preliminary experiments indicate effectiveness in zebrafish. In Cyprinus carpio, we detail our progress at creating EGI, assessing wild type genetic diversity of candidate EGI carp promoters, and our pipeline for attaining year round transgenesis .
EGI relies on targeting genetically conserved promoter regions in wild populations. To assess wild carp genetic diversity, we sequenced four targetable promoters (shha, gata5_1, gata5_2, and ern1) from 250 fish sample s from 6 different environments (5 lakes and 1 domesticated lab strain). Results show that all surveyed populations shared a few highly conserved targetable haplotypes for each promoter. To begin the steps of EGI in C. carpio, a facility for year-round spawning was produced . One year old carp were introduced to an indoor flow through facility kept at 24C 16L/8D for 3 months. Then, fish were acclimated over 8 days to 16C 12L/12D for periods ranging from 3-21 months. Finally, mature fish were warmed to 24C 16L/8D over the course of a month and induced to spawn with Ovaprim injections . After in vitro fertilization, t ransgenesis was performed in C. carpio embryos by co- injecting tol2 mRNA and a plasmid containing GFP driven by the carp beta-actin promoter. Experiments comparing deadhesion solutions (milk, bentonite, and tannic acid) and their effects on survival were evaluated. Females were spawned successfully even when held at 16C for 21 months. GFP-positive transgenic fish were successfully produced . It was demonstrated that stripped oocytes can be kept at room temperature for up to 5 hours before ensuing embryo survival drops below 30% . It was found that a 3 minute 500 ppm tannic acid treatment was the most effective for reducing egg adhesion and resulted in the highest embryo survival after microinjection .