The mutagenic chain reaction: a method for autocatalyic gene dissemination

诱变链式反应：一种自催化基因传播的方法

• 批准号: 10211352
• 负责人: ETHAN BIER
• 金额: $ 32.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211352
• 关键词: Alleles; Anopheles Genus; Antibiotic Resistance; Antibodies; Antimalarials; Bacteria; Blastoderm; CRISPR gene drive; Child; Chromosomes; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Combination Drug Therapy; Complementary DNA; Complex; Culicidae; DNA; DNA Repair; Development; Disease; Drosophila genus; Drug resistance; Elements; Embryo; Event; Female; Foundations; Future; Genes; Genetic; Genome; Genomics; Germ Cells; Government; Grant; Guide RNA; Health; Homologous Gene; Human; Image; Immunize; Individual; Inherited; Insect Vectors; Insecta; Insecticide Resistance; Insecticides; Intervention; Laboratories; Malaria; Mammals; Measures; Mediating; Methods; Mosaicism; Mus; Mutagenesis; Mutate; Parasite resistance; Parasites; Pathway interactions; Performance; Population; Poverty; Poverty Areas; Prevalence; Process; Prokaryotic Cells; Reaction; Reproduction; Resistance; Sand Flies; Sanitation; Scourge; Site; Source; Sterility; System; Target Populations; Technology; Testing; Transgenes; Transgenic Organisms; Tsetse Flies; Update; Vector-transmitted infectious disease; Virus; World Health Organization; base; blastomere structure; blocking factor; combat; cost; design; ds-DNA; endonuclease; experimental study; flexibility; fly; food insecurity; functional restoration; gene drive system; genetic element; genetic technology; genetic variant; genomic locus; global health; imaging modality; loss of function; malaria transmission; mutagenic chain reaction; next generation; novel; pathogen; prototype; repaired; tool; trait; transmission process; vector; welfare

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.

经过十年的重大进步，全球根除议程已停滞不前，应 部分是抗杀虫剂蚊子和耐药疟原虫的出现。 世界卫生组织和其他人呼吁制定新策略以帮助失败 这种毁灭性的疾病会感染超过200万人，每年杀死超过40万人，主要是 贫困地区的幼儿。基因驱动器可以偏向所需特征的遗传，提供了一本小说 并有希望消除引起昆虫媒介的疾病，或者将其免疫的有希望的策略 病原体。这种超级孟德尔CRIPSR基因驱动系统编码双方转基因盒 由CAS9核酸内切酶和引导RNA（GRNA）组成，该指导在基因组上引导DNA裂解 插入部位。在生殖细胞中，这种靶向切割同源染色体会导致复制 通过同源性维修在裂解部位的驱动元件，几乎所有后代 继承驱动元件及其货物。 我的小组为开发第一个基于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。我们还开创了旨在偏见的等位基因驱动系统 在一个单独的遗传基因座上的偏爱等位基因变体的继承。此外，我们已经开发了， 经过广泛测试，两种类型的自复印驱动器中和系统，两种都带有grnas，但没有 CAS9的来源。 ERACRS删除并替换基因驱动器，而E-Chacrs在突变时会复制自己 并灭活基因驱动的Cas9转基因。果蝇和 蚊子表明，高效的基因驱动器迅速通过目标种群传播，并且 ERACR和E-CHACR可以可靠地替换（ERACR）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个灵活的两个组件（拆分驱动器或CHACR）系统，可以 将基因转换（或黑客攻击）转换为单个全驱动系统。分裂和完整的驱动器元素是 插入对于生存力或繁殖必不可少的基因中，还携带靶向基因的重新编码cDNA 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现象中受益匪浅 并被称为致命/无菌镶嵌物，它主要消除了功能丧失等位基因（错误） 目标基因由不精确的DNA维修事件而不是预期的复制事件产生。接下来，我们会的 开发和测试下一代ERACR和E-CHACRS能够消除或停止我们的重新编码驱动器，也 测试一个自限制的驱动系统，该系统缓慢靶向CAS9进行诱变。与这些驱动实验并行 我们将使用一组唯一的基于图像的遗传来深入研究驱动过程的机制和时机 我们开发的要素。我们预计智力的进步和可改变游戏的进步 这些研究的技术将有助于解决人类健康中的关键全球挑战。