The mutagenic chain reaction: a method for autocatalyic gene dissemination

 描述(由申请人提供)： 对不同先锋物种基因组序列数据的加速获取增加了对新的遗传工具的需求，以探索先锋生物的基因功能。在控制昆虫病媒和动植物害虫方面，另一个尚未得到满足的重要需求是建立一个系统，将效应器转基因分散到这些物种的野生种群中。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，称为突变链式反应(MCR)，它可以在一个步骤中将杂合等位基因转换为纯合状态。由于MCR在生殖系中以及在体细胞中都有效地发挥作用，这项技术也广泛适用于 通过一种极端形式的驱动力在一群生物体内或在一个生物体内的细胞内传播遗传因素。在这个应用中，我们描述了MCR方法，并建议在果蝇中描述这一新的遗传系统。MCR方法基于由三个组成部分组成的结构(MCR元件)：1)编码细菌Cas9内切酶的基因，2)针对Cas9切割到特定基因组位置的引导RNA(GRNA)基因，以及3)同源臂侧翼并直接毗邻gRNA定向切割位点。一个MCR携带的Cas9和gRNA(S)导致另一个等位基因上基因组DNA的切割，侧翼的同源臂导致该构建物通过同源定向修复复制到基因组中，从而使MCR构建物纯合。初步实验表明，MCR在体细胞和生殖细胞中都是高效的(&gt；95%)。因此，这样的元素应该在种群中呈指数级传播，最初在每一代人中翻一番。MCR元件转化相反的等位基因并通过种系有效传播的能力在许多重要应用中都具有重要价值，包括先锋生物体的单步突变、加速所有生物体的基因操作、提供一种有效的遗传驱动形式以帮助在害虫种群中传播转基因(例如在蚊子中抗击疟疾)，以及潜在地在体内广泛传递基因结构以对抗艾滋病毒、癌症和基因治疗策略。我们还建议开发工具来帮助限制扩散或在必要时消除MCR元件，例如用于自催化链式反应(ERACR)逆转的元件。ERACR元件携带两个指导MCR插入位点两侧基因组序列切割的gRNA，以及毗邻这些切割位点的同源臂，但不携带Cas9内切酶的来源。ERACRs应该能够在携带目标MCR元件的种群中呈指数级传播，但对缺乏Cas9来源的野生生物应该没有影响。在这个方案中，我们将表征影响MCR元件传播的参数，测量MCR的靶效应并开发相反的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.