The mutagenic chain reaction: a method for autocatalyic gene dissemination

经过十年的重大进展，全球消灭疟疾议程已经停滞， 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， 这种毁灭性的疾病每年感染超过200万人，导致超过40万人死亡，其中主要是 贫困地区的儿童。基因驱动，可以偏向所需性状的遗传，提供了一个新的 和有前途的战略，无论是消除疾病引起的昆虫载体，或免疫他们对 病原体这种基于超孟德尔CRIPSR的基因驱动系统编码二分转基因盒 由Cas9内切核酸酶和指导RNA（gRNA）组成，其指导基因组DNA切割。 插入部位。在生殖细胞中，同源染色体的这种定向切割导致复制 通过同源性定向修复在切割位点的驱动元件，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， 在一个单独的基因座上遗传一个有利的等位基因变体。此外，我们还开发了 经过广泛测试，两种类型的自我复制驱动器中和系统，两者都携带gRNA，但没有 Cas9的来源ERACR删除和替换基因驱动，而e-CHACR在突变时复制自己 以及使基因驱动器上携带的Cas9转基因失活。在苍蝇中进行的小种群笼实验， 蚊子已经证明，高效的基因驱动在目标人群中迅速传播， ERACR和e-CHACR可以可靠地替换（ERACR）或停止（e-CHACR）基因驱动元件。 在这项资助中，我们建议首先开发一种灵活的双组分（分离驱动或CHACR）系统， 被基因转换（或黑客攻击）成一个单一的全驱动系统。分离和全驱动元件是 插入生存力或繁殖所必需的基因，并且还携带靶基因的重新编码的cDNA 来恢复这些位点的功能。这些重新编码的系统从我们发现的一个现象中受益匪浅 并被称为致死/不育嵌合体，其主要消除基因组中的功能丧失等位基因（错误）。 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。接下来我们就 开发和测试下一代ERACR和e-CHACR，能够消除或停止我们的记录驱动器， 测试缓慢靶向Cas9进行诱变的自限性驱动系统。在这些驱动实验的同时， 我们将使用一套独特的基于图像的遗传算法， 我们开发的元素。我们预计，知识的进步和可实施的改变游戏规则 这些研究所产生的技术将为解决人类健康方面的重大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.