Department of Life Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Facility for Imaging by Light Microscopy, NHLI, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Sci Rep. 2018 Sep 3;8(1):13125. doi: 10.1038/s41598-018-31433-2.
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.
理性设计的生殖隔离屏障在动植物物种中的应用具有多种生物技术应用的潜力，例如创建遗传防火墙、控制基因驱动或新型种群替换和遗传控制抑制策略。然而，迄今为止，还没有实验数据探索多细胞生物中这一概念。在这里，我们通过结合基于 CRISPR 的基因组编辑和相同基因座的转录激活（CRISPRa），在模式生物黑腹果蝇中检查了在后生动物中构建人工生殖隔离的要求。我们将 13 个单引导 RNA（sgRNA）靶向 7 个进化保守基因的启动子，并使用 11 个驱动子进行了错误激活筛选。我们确定了 eve 基因座的显性致死激活因子，发现它们通过强烈激活 eve 超出其天然时空背景而破坏发育。我们使用相同的 sgRNA 集合通过基因组编辑分离出保护性 INDEL，这些 INDEL 使这些基因座能够抵抗激活而不干扰靶基因功能。当组合使用这组遗传元件时，我们发现使用这种方法可以实现大多数应用所必需的完全合成致死性。然而，我们的结果表明，dCas9 表达的水平和范围、遗传隔离的程度以及对苍蝇适应性的影响之间存在着陡峭的权衡。我们提出的这种遗传工程策略允许创建单个或多个生殖隔离屏障，并可应用于其他多细胞生物，如病媒或具有经济重要性的转基因生物。
