The mutagenic chain reaction: a method for autocatalyic gene dissemination

在十年的重大进展之后，全球根除疟疾议程停滞不前， 部分原因是抗药性蚊子和抗药性疟疾寄生虫的加速出现。 世界卫生组织和其他组织呼吁制定新的战略来帮助战胜 这种毁灭性的疾病感染了200多万人，每年导致40多万人死亡，主要是 贫困地区的幼儿。基因驱动可以偏向所需性状的遗传，这提供了一种新的 有希望的策略要么是消灭致病媒介，要么是对它们进行免疫 病原体。这种基于超级孟德尔CRIPSR的基因驱动系统编码两部分转基因盒带 由Cas9内切酶和引导DNA在基因组上切割的引导RNA(GRNA)组成 插入位置。在生殖细胞中，这种同源染色体的定向切割会导致复制 裂解部位的驱动元件通过同源定向修复，导致几乎所有的后代 继承驱动元件及其货物。 我的团队为开发第一个基于CRIPSR的基因驱动(或主动遗传)系统做出了贡献 苍蝇、蚊子、哺乳动物和细菌。我们还开创了等位基因驱动系统，旨在偏向 在单独的遗传座位上遗传一个受欢迎的等位基因变异。此外，我们还开发了，和 经过广泛测试的两种类型的自复制驱动器中和系统，都带有gRNA，但没有 Cas9的来源。ERACR删除和取代基因驱动，而e-CHACR在突变的同时复制自己 灭活Cas9转基因进行了基因驱动。小种群笼养苍蝇和 蚊子已经表明，高效的基因驱动在目标人群中迅速传播，而且 ERACRs和e-CHACRs可以可靠地取代(ERACRs)或停止(e-CHACRs)基因驱动元件。 在这笔赠款中，我们首先建议开发一种灵活的双组件(拆分驱动或CHACR)系统，该系统可以 被基因转换(或被黑客攻击)为单一的全驱动系统。分离式和全驱动元件包括 插入对生存或繁殖至关重要的基因，还携带目标基因的重新编码的cDNA 以恢复这些基因座的功能。这些重新编码的系统从我们发现的一种现象中受益匪浅 并被称为致死/不育嵌合体，它主要消除了 由不精确的DNA修复事件而不是预期的复制事件产生的靶基因。接下来，我们将 开发和测试下一代ERACR和e-CHACR，能够消除或停止我们的重新编码驱动器，以及 测试一个自限驱动系统，该系统缓慢地以Cas9为目标进行诱变。在进行这些驾驶实验的同时， 我们将使用一组独特的基于图像的基因来深入研究驱动过程的机制和时间 我们开发的元素。我们预计，智力的进步和可实施的游戏规则改变 来自这些研究的技术将对解决人类健康方面的关键全球挑战作出重要贡献。

Following a decade of significant strides forward, the global malaria eradication agenda has stalled, due in part to the accelerating emergence of insecticide-resistant mosquitoes and drug-resistant malarial parasites. The World Health Organization and others have called for the development of new strategies to help defeat this devastating disease that infects over 2 million people and killing over 400,000 annually, predominantly young children in impoverished regions. Gene-drives, which can bias inheritance of desired traits, offer a novel and promising strategy either to eliminate disease causing insect vectors, or to immunize them against pathogens. Such super-Mendelian CRIPSR-based gene-drive systems encode bipartite transgenic cassettes consisting of the Cas9 endonuclease and a guide RNA (gRNA), which directs DNA cleavage at the genomic site of insertion. In reproductive cells, such targeted cutting of the homologous chromosome results in copying the drive element at the cleavage site through homology directed repair, resulting in nearly all progeny inheriting the drive element and its cargo. My group has contributed to developing the first CRIPSR-based gene drive (or active genetic) systems in flies, mosquitoes, mammals, and bacteria. We also pioneered allelic-drive systems designed to bias inheritance of a favored allelic variant at a separate genetic locus. In addition, we have developed, and extensively tested, two types of self-copying drive neutralizing systems, both of which carry gRNAs, but no source of Cas9. ERACRs delete and replace gene-drives, while e-CHACRs copy themselves while mutating and inactivating the Cas9 transgene carried on a gene-drive. Small population cage experiments in flies and mosquitoes have shown that highly efficient gene-drives rapidly spread through target populations, and that ERACRs and e-CHACRs can reliably replace (ERACRs) or halt (e-CHACRs) a gene-drive element. In this grant, we propose first to develop a flexible two-component (split-drive or CHACR) system that can be genetically converted (or hacked) into a single full-drive system. The split and full drive elements are inserted into genes essential for viability or reproduction, and also carry recoded cDNAs of the targeted genes to restore function of those loci. These recoded systems benefit greatly from a phenomenon we discovered and refer to as lethal/sterile mosaicism, which dominantly eliminates loss-of-function alleles (mistakes) in the target gene generated by imprecise DNA repair events rather than the intended copying event. Next, we will develop and test next-generation ERACR and e-CHACRs able to eliminate or halt our recoded-drives, and also test a self-limiting drive system that slowly targets Cas9 for mutagenesis. In parallel to these drive experiments, we will delve into the mechanisms and timing of the drive process using a unique set of image-based genetic elements we have developed. We anticipate that the intellectual advances and implementable game-changing technologies from these studies will contribute importantly to solving critical global challenges in human health.