CRISPR/Cas9-Mediated Generation and Characterization of an Ent2*/CyO Drosophila melanogaster Strain.
In this study, a CRISPR/Cas9-based genome-editing approach was used to introduce mutations in the equilibrative nucleoside transporter 2 (Ent2) gene in Drosophila melanogaster. Guide RNAs targeting the coding region of Ent2 were designed and co-injected with Cas9 mRNA into w1118 embryos. Mutant alleles were identified by Sanger sequencing and maintained as a stable Ent2*/CyO heterozygous line using a balancer chromosome. Subsequently, we evaluated body weight, climbing ability, survival rate, and the activities of superoxide dismutase (SOD) and catalase (CAT) in fruit flies at 22 °C and 25 °C, respectively. The results indicate that at both 22 °C and 25 °C, the body length and weight of Ent2*/CyO fruit flies were significantly reduced compared to the w1118, and their development was delayed. At 22 °C, the overall lifespan of Ent2*/CyO flies was slightly longer than that of the w1118, whereas at 25 °C, no significant difference was observed. Regarding locomotor ability, the climbing performance of heterozygous flies was significantly lower than that of the w1118 at both temperatures, with males being more severely affected. In addition, the antioxidant enzyme activities of CAT and SOD in Ent2*/CyO fruit flies were significantly reduced, indicating a clear impairment of antioxidant capacity. These results describe the phenotypic profile of a CRISPR-generated Ent2 mutant line and demonstrate the feasibility of combining genome editing with balancer chromosome strategies in Drosophila. This study provides a methodological framework and a genetic resource for future investigations of genes associated with metabolism and environmental responses.