[论文解读] RPM-Drive: A robust, safe, and reversible gene drive system that remains functional after 200+ generations

Floyd A. Reed, Todd G. Aquino-Michaels+3 more|arXiv (Cornell University)|Jun 13, 2018

CRISPR and Genetic Engineering参考文献 38被引用 6

一句话总结

本研究证明，果蝇（Drosophila melanogaster）中基于合成性下位性抑制的RPM-Drive基因驱动系统在超过200代后仍保持完全功能性和遗传稳定性。该系统利用核糖体蛋白基因的单倍体不足特性，并内置遗传安全机制，自然选择能有效清除破坏性突变，使其成为一种强大、可逆且地理受限的遗传害虫管理解决方案。

ABSTRACT

Despite the advent of several novel, synthetic gene drive mechanisms and their potential to one-day control a number of devastating diseases, among other applications, practical use of these systems remains contentious and risky. In particular, there is little in the way of empirical evidence of the long-term robustness of these synthetic systems against mutational breakdown. Rather, most existing systems are either known or predicted to be susceptible to rapid inactivation, though methodological designs continue to be refined. Here we evaluate a currently existing synthetic, underdominance-based gene drive system 200+ generations after it was first established in a laboratory colony of Drosophila melanogaster. Not only do we find that the system is still functioning as designed, we also show evidence that disruptions to the genetic construct are highly likely to be removed by natural selection, contributing to the system's robust, long-term stability. This stability appears to be a result of a fundamental relationship between ribosomal proteins (a novel target of the system) and natural cellular defenses that protect against cancer development. As far as we are aware, this is the longest continually functioning synthetic gene drive system thus verified, making it highly appropriate for additional research into its eventual suitability for field trials. Due to inherent properties of this gene drive, it is also likely to be adaptable for use in many different species. The insect lines established and used to test this system have been deposited at a Drosophila stock center, and are available to labs for further, independent testing.

研究动机与目标

• 评估基因驱动系统在200代以上的长期功能稳定性。
• 评估RPM-Drive在实验种群中对突变崩溃的抗性。
• 研究自然选择在清除基因驱动构建体中破坏性突变方面的角色。
• 通过工程化遗传安全机制，测试该系统在可逆性和地理隔离方面的表现。
• 基于其机制设计，评估RPM-Drive在不同物种间的可移植性与适用性。

提出的方法

• 在20个独立培养管中，以每管约100只成虫的规模，将果蝇（Drosophila melanogaster）的RPM-Drive品系连续传代超过200代。
• 在RPM-Drive纯合子与对照‘野生型’品系之间进行互交实验，以评估孟德尔遗传规律和驱动效率。
• 通过遗传杂交模拟GAL4转录激活因子的丢失（安全机制），以模拟意外释放的情景。
• 利用RFP和GFP标记对F1和F2后代进行基因分型，以确认基因型比例。
• 比较核糖体蛋白（Rp）剂量水平（1、2或3个功能拷贝），以评估基因剂量失衡对适合度的影响。
• 通过分析后代存活率和标记表达，推断对突变基因型的选择作用，验证系统稳定性。

实验结果

研究问题

• RQ1 RPM-Drive系统在连续传代200代以上后是否仍保持功能性和可遗传性？
• RQ2 随着时间推移，基因驱动构建体中的破坏性突变在多大程度上被自然选择清除？
• RQ3 核糖体蛋白单倍体不足机制如何促进该系统长期稳定性？
• RQ4 在存在基因驱动的情况下，核糖体蛋白剂量（1、2或3个拷贝）的差异对适合度有何影响？
• RQ5 内置的GAL4/Cy-安全机制是否能有效在意外杂交后使驱动失活，从而确保可逆性和地理隔离？

主要发现

• 在200代以上传代后，F2代中RPM-Drive系统维持了孟德尔基因型比例（1:2:1），表明其功能持续且遗传忠实度高。
• 对基因构建体的破坏（如GAL4转录激活因子的丢失）被自然选择迅速清除，GAL4/Cy-安全机制在不到一代内将驱动活性降至接近零。
• 仅携带一个功能性RpL14拷贝（剂量=1）的个体相比具有两个或三个拷贝的个体，表现出显著降低的适合度，证实了单倍体不足机制。
• 该系统完全可逆：当GAL4系统通过独立分配丢失时，驱动效应迅速被抑制，防止了意外扩散。
• RPM-Drive的长期稳定性归因于核糖体蛋白功能所施加的进化约束，该功能与细胞防御癌症机制相关，从而增强了系统的鲁棒性。
• 携带RPM-Drive的果蝇品系已存入布卢明顿果蝇资源中心（Bloomington Drosophila Stock Center），支持独立验证并促进更广泛的研究应用。