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

基本信息

批准号：
10614935
负责人：
ETHAN BIER
金额：
$ 32.39万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
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/10614935
关键词：
Acceleration Alleles Anopheles Genus Antibiotic Resistance Antibodies Antimalarials Bacteria Binding Blastoderm CRISPR gene drive Child Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Combination Drug Therapy Complementary DNA Complex Culicidae DNA DNA Double Strand Break DNA Repair DNA cassette Development Disease Drosophila genus Drug resistance Elements Embryo Event Female Foundations Future Genes Genetic Genome Genomics Germ Cells Government Grant Guide RNA Health Heterozygote 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 Persons Phlebotominae Population Poverty Poverty Areas Prevalence Process Prokaryotic Cells Reaction Reproduction Resistance Sand Flies Sanitation Scourge Site Source Specific qualifier value Sterility System Target Populations Technology Testing Transgenes Tsetse Flies Update Vector-transmitted infectious disease Virus Visualization World Health Organization blastomere structure blocking factor combat cost design embryo cell 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 pragmatic implementation prototype rational design repaired tool trait transmission process vector welfare

项目摘要

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.

经过十年的重大进展，全球消除疟疾议程陷入停滞，原因是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战略来帮助战胜这种毁灭性的疾病每年感染超过 200 万人并导致超过 40 万人死亡，其中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，提供了一种新颖的方法消除致病昆虫媒介或对它们进行免疫的有前景的策略病原体。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 核酸内切酶和引导 RNA (gRNA) 组成，引导 DNA 在基因组上进行切割插入位点。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定向修复切割位点的驱动元件，产生几乎所有后代继承驱动元素及其货物。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，旨在偏向在单独的基因位点遗传偏好的等位基因变异。此外，我们还开发了经过广泛测试，两种类型的自复制驱动器中和系统都携带 gRNA，但没有 Cas9的来源。 ERACR 删除并替换基因驱动，而 e-CHACR 在变异时复制自身并使基因驱动的 Cas9 转基因失活。果蝇小群体笼实验蚊子已经表明，高效的基因驱动在目标人群中迅速传播，并且 ERACR 和 e-CHACR 可以可靠地替换 (ERACR) 或停止 (e-CHACR) 基因驱动元件。在这笔拨款中，我们建议首先开发一种灵活的双组件（分体驱动或 CHACR）系统，该系统可以被基因转换（或破解）为单个全驱动系统。分体式和全式驱动元件是插入到生存或繁殖所必需的基因中，并且还携带目标基因的重新编码的 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称为致命/不育嵌合体，它主要消除了基因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目标基因。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，并且测试一个自限性驱动系统，该系统缓慢地针对 Cas9 进行诱变。在进行这些驱动实验的同时，我们将使用一组独特的基于图像的遗传技术深入研究驱动过程的机制和时间安排我们开发的元素。我们预计智力进步和可实施的游戏规则改变这些研究的技术将为解决人类健康方面的关键全球挑战做出重要贡献。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly.

通过染色体水平基因组组装揭示了侵袭性疟疾载体史氏按蚊的隐藏基因组特征。

DOI：
发表时间：
2021-02-10
期刊：
影响因子：
5.4
作者：
Chakraborty, Mahul;Ramaiah, Arunachalam;Adolfi, Adriana;Halas, Paige;Kaduskar, Bhagyashree;Ngo, Luna Thanh;Jayaprasad, Suvratha;Paul, Kiran;Whadgar, Saurabh;Srinivasan, Subhashini;Subramani, Suresh;Bier, Ethan;James, Anthony A;Emerson, J J
通讯作者：
Emerson, J J

Development of an anti-Pfs230 monoclonal antibody as a Plasmodium falciparum gametocyte blocker.

开发抗 Pfs230 单克隆抗体作为恶性疟原虫配子体阻断剂。