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: 9009589
• 负责人: ETHAN BIER
• 金额: $ 30.61万
• 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 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9009589
• 关键词: Acceleration; Address; Adverse effects; Agriculture; Alleles; Animals; Beryllium; CRISPR/Cas technology; Cells; Chromosomes; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Culicidae; Data; Development; Disease; Disease Vectors; Drosophila genus; Drosophila melanogaster; Elements; Embryo; Frequencies; Gene Targeting; Generations; Genes; Genetic; Genome; Genome engineering; Genomic DNA; Genomics; Guide RNA; HIV; HIV therapy; Human body; Indium; Injection of therapeutic agent; Introns; Lead; Malaria; Malignant Neoplasms; Measures; Medical; Methods; Mutagenesis; Mutation; Organism; Plants; Population; Protocols documentation; Reaction; Resistance; Risk; Safety; Site; Somatic Cell; Source; System; Technology; Transgenes; Transgenic Organisms; arm; base; cancer therapy; combat; endonuclease; gene function; gene therapy; genetic element; genetic manipulation; genome editing; genome sequencing; insect disease; novel; public health relevance; repaired; research study; tool; vector

 DESCRIPTION (provided by applicant): The accelerating acquisition of genome sequence data in diverse pioneer species has heightened the need for new genetic tools to explore gene function in pioneer organisms. Another important unmet need in control insect disease vectors and animal and plant pests is a system for dispersing effector transgenes into wild populations of these species. To help address these needs, we have developed a new method based on the CRISPR/Cas9 genome editing system referred to as the Mutagenic Chain Reaction (MCR), which results in the converts heterozygous alleles to the homozygous state in a single step. Because MCR acts efficiently in the germline as well in somatic cells, this technology is also broadly applicable to dispersing genetic elements via an extreme form of drive within a population of organisms or within cells within an organism. In this application we describe the MCR method and propose to characterize this novel genetic system in Drosophila. The MCR method is based on constructs (MCR elements) comprised of three constituents: 1) a gene encoding the bacterial Cas9 endonuclease, 2) a guide-RNA (gRNA) genes that targets Cas9 cleavage to specific genomic sites, and 3) homology arms flanking and directly abutting the gRNA directed cut sites. Cas9 and gRNA(s) carried by an MCR lead to cleavage of the genomic DNA on the other allele and the flanking homology arms result in that construct being copied into the genome via homology directed repair thereby making the MCR construct homozygous. Preliminary experiments indicate that MCR is highly efficient in both somatic and germline cells (>95%). Thus, such elements should spread exponentially through a population, initially doubling at each generation. The ability of MCR elements convert the opposing allele and to spread efficiently via the germline should be of significant value in a broad variety important applications including single step mutagenesis in pioneer organisms, accelerating genetic manipulations in all organisms, providing a potent form of genetic drive to help spread transgenes throughout populations of pest species (e.g., in mosquitoes to combat malaria), and potentially for broad delivery of genetic constructs within the body to combat diseases such as HIV, cancer, and gene therapy strategies. We also propose to develop tools to help limit the spread or eliminate MCR elements if necessary such as an Element for Reversion of the Autocatalytic Chain Reaction (ERACR). ERACR elements carry two gRNAs directing cleave of genomic sequences flanking an MCR insertion site, as well as homology arms abutting these cleavage sites, but do not carry a source of Cas9 endonuclease. ERACRs should be able to spread exponentially throughout a population carrying the targeted MCR element but should have no effect in wild-type organisms lacking a source of Cas9. In this proposal, we will characterize parameters influencing the spread of MCR elements, measure off target effects of MCR and develop opposing ERACR elements and conduct competition experiments between MCR and ERACR elements in populations.

 描述（由申请人提供）： 潜水先驱物种中基因组序列数据的加速习得增强了对探索先锋生物中基因功能的新遗传工具的需求。控制绝缘疾病媒介以及动物和植物害虫的另一个重要需求是将效应子翻译分散成这些物种的野生种群的系统。为了帮助满足这些需求，我们开发了一种基于CRISPR/CAS9基因组编辑系统（称为诱变链反应（MCR））的新方法，该系统在单一步骤中导致将杂合等位基因转换为纯合状态。由于MCR在体细胞中也有效地在种系中起作用，因此该技术也广泛适用于 通过在组织中或组织内部的细胞内部或细胞内部的极端驱动形式分散遗传因素。在此应用中，我们描述了MCR方法和建议，以表征果蝇中这种新型遗传系统的表征。 MCR方法基于三个构建体的构建体（MCR元素）：1）编码细菌Cas9核酸酶的基因，2）靶向Cas9裂解至特定基因组位点的指导RNA（GRNA）基因，以及3）侧翼和直接直接将GRNA定向的切割位点直接。通过MCR携带的Cas9和GRNA导致基因组DNA在另一个等位基因上的裂解，并且侧翼同源性臂导致该构建体通过同源性修复复制到基因组中，从而使MCR构造纯合子。初步实验表明，MCR在体细胞和种系细胞中均高效（> 95％）。这就是这样的要素应通过人口成倍扩散，最初在每一代人中翻了一番。 The ability of MCR elements convert the opposing allele and to spread efficiently via the germline should be of significant value in a broad variety important applications including single step mutagenesis in pioneer organisms, accelerating genetic manipulations in all organisms, providing a potential form of genetic drive to help spread translations throughout populations of pest species (e.g., In mosquitoes to combat malaria), and potentially for broad delivery of genetic constructs within the抗击艾滋病毒，癌症和基因治疗策略等疾病的身体。我们还建议开发工具，以帮助限制扩散或消除MCR元素，例如用于自催化链反应（ERACR）的元素。 ERACR元素携带两个指导MCR插入位点两侧的基因组序列，以及依靠这些清洁位点的同源臂，但不带Cas9核酸内切核酸酶的来源。 ERACRS应该能够在携带靶向MCR元件的人群中呈指数分散，但对缺乏CAS9来源的野生型生物没有影响。在此提案中，我们将表征参数会影响MCR元素的传播，测量MCR的目标影响并发展相反的ERACR元素，并在人群中MCR和ERACR元素之间进行竞争实验。