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.

冷暴露后的存活与繁殖能力，在所有生物界中均具有重要意义。然而，即便在黑腹果蝇（Drosophila melanogaster）这类成熟的经典遗传模式生物中，被证实参与耐寒调控的基因仍寥寥无几。冷暴露后，Frost（Fst）基因的转录本积累水平显著升高，使其成为耐寒调控相关的优良候选基因。然而，尽管已开展了大量RNA干扰（RNAi）敲降分析，却始终未能证实Fst在耐寒过程中发挥任何功能。CRISPR是一种可实现基因完全敲除的高效技术，相较于GAL4-UAS RNA干扰系统，其脱靶效应发生率更低。我们借助CRISPR介导的同源重组技术构建了Fst基因的纯合敲除等位品系，通过这些品系发现，FST蛋白在冷暴露后维持雌性繁殖力的过程中不可或缺，但FST并未直接参与冷暴露后的存活调控。FST的mRNA在马氏管（Malpighian tubules）中积累，且FST蛋白属于高度无序蛋白，带有一段推测的细胞分泌信号肽。后续仍需开展研究，以明确FST是否从马氏管分泌，并在冷暴露后与雌性生殖组织直接相互作用；抑或是通过参与其他修复/恢复过程，间接改变繁殖相关的能量分配，从而发挥其功能。