The mutagenic chain reaction: a method for autocatalyic gene dissemination

 描述（由申请人提供）： 在不同的先驱物种的基因组序列数据的加速收购提高了需要新的遗传工具，以探索基因功能的先驱生物。在控制昆虫疾病媒介和动物和植物害虫中另一个重要的未满足的需求是用于将效应转基因分散到这些物种的野生群体中的系统。为了帮助解决这些需求，我们开发了一种基于CRISPR/Cas9基因组编辑系统的新方法，称为诱变链式反应（MCR），它可以在一个步骤中将杂合等位基因转化为纯合状态。由于MCR在生殖系以及体细胞中有效地起作用，因此该技术也广泛适用于 通过极端形式的驱力在生物群体内或在生物体内的细胞内分散遗传元素。在这个应用程序中，我们描述了MCR方法，并建议在果蝇中描述这种新的遗传系统。MCR方法基于由三种成分组成的构建体（MCR元件）：1）编码细菌Cas9内切核酸酶的基因，2）靶向Cas9切割至特定基因组位点的向导RNA（gRNA）基因，和3）侧接并直接邻接gRNA定向切割位点的同源臂。由MCR携带的Cas9和gRNA导致另一个等位基因上的基因组DNA的切割，并且侧翼同源臂导致该构建体通过同源定向修复被复制到基因组中，从而使MCR构建体纯合。初步实验表明，MCR在体细胞和生殖细胞中都是高效的（>95%）。因此，这些元素应该在种群中呈指数级传播，最初每一代都翻一番。MCR元件转化相对等位基因并通过种系有效传播的能力在广泛的各种重要应用中具有重要价值，包括先驱生物体中的单步诱变，加速所有生物体中的遗传操作，提供有效形式的遗传驱动以帮助在整个害虫物种群体中传播转基因（例如，在蚊子中对抗疟疾），并有可能在体内广泛传递基因构建体，以对抗艾滋病毒，癌症和基因治疗策略等疾病。 我们还建议开发工具来帮助限制MCR元素的传播或在必要时消除MCR元素，例如自催化链式反应逆转元素（ERACR）。ERACR元件携带指导MCR插入位点侧翼的基因组序列的切割的两个gRNA，以及邻接这些切割位点的同源臂，但不携带Cas9内切核酸酶的来源。ERACR应该能够在携带靶向MCR元件的群体中指数传播，但在缺乏Cas9来源的野生型生物体中应该没有影响。在这项提案中，我们将表征影响MCR元件传播的参数，测量MCR的脱靶效应，开发相反的ERACR元件，并在种群中进行MCR和ERACR元件之间的竞争实验。

 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.