Data from: CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure

The ability to survive and reproduce after cold exposure is important in all kingdoms of life. However, even in a sophisticated genetic model system like Drosophila melanogaster, few genes have been identified as functioning in cold tolerance. The accumulation of the Frost (Fst) gene transcript increases after cold exposure, making it a good candidate for a gene that has a role in cold tolerance. However, despite extensive RNAi knockdown analysis, no role in cold tolerance has been assigned to Fst. CRISPR is an effective technique for completely knocking down genes, and less likely to produce off-target effects than GAL4-UAS RNAi systems. We have used CRISPR-mediated homologous recombination to generate Fst null alleles, and these Fst alleles uncovered a requirement for FST protein in maintaining female fecundity following cold exposure. However, FST does not have a direct role in survival following cold exposure. FST mRNA accumulates in the Malpighian tubules, and the FST protein is a highly disordered protein with a putative signal peptide for export from the cell. Future work is needed to determine whether FST is exported from the Malpighian tubules and directly interacts with female reproductive tissues post-cold exposure, or if it is required for other repair/recovery functions that indirectly alter energy allocation to reproduction.

在所有生物界中，冷暴露后存活与繁殖的能力至关重要。然而，即便在黑腹果蝇（Drosophila melanogaster）这类成熟的经典遗传模式系统中，被证实参与耐寒调控的基因仍寥寥无几。冷暴露会诱导Frost（Fst）基因转录本积累，使其成为耐寒调控候选基因的理想对象。但即便经过大规模RNA干扰（RNAi）敲降分析，学界仍未明确Fst在耐寒过程中的功能。成簇规律间隔短回文重复序列（CRISPR）是实现基因完全敲除的高效技术，且相较于GAL4-UAS RNAi系统，其脱靶效应的发生概率更低。我们利用CRISPR介导的同源重组技术构建了Fst纯合敲除等位基因，通过该等位基因的表型分析发现，FST蛋白在雌性个体冷暴露后的繁殖力维持过程中不可或缺。但FST并未直接参与冷暴露后的存活调控。FST mRNA在马尔皮基氏小管（Malpighian tubules）中富集，且该蛋白属于高度无序蛋白，带有潜在的细胞分泌信号肽。后续仍需开展研究，以明确FST是否从马尔皮基氏小管分泌，并在冷暴露后直接与雌性生殖组织相互作用，抑或其通过参与其他修复/恢复过程，间接调控繁殖相关的能量分配。