Improving the efficiency and control of CRISPR/Cas9 gene drive systems

许多重要的人类病原体，包括那些负责疟疾，登革热，黄热病，寨卡病毒，西 尼罗河热和基孔肯雅热都是由蚊子传播的。一个有效的“基因驱动”系统可以 从根本上改变我们控制这些疾病媒介的策略， 将基因工程改造的等位基因导入载体群体中。最近开发的CRISPR/Cas9基因驱动 (CGD)系统承诺了一个高度适应性的机制，通过转换 驱动子的杂合子构建成生殖系中的纯合子。然而，目前尚不清楚如何 这种机制在野生种群中是有效的，在那里遗传异质性和突变可能 产生抵抗驱动机制的等位基因。重要的是，这些抗性等位基因将被 当通过非同源末端连接（NHEJ）修复切割时，由驱动器本身产生。的目标 该建议是开发具有降低的抗性等位基因形成速率的CGD构建体， 实验量化了对大笼中抗性等位基因进化重要的因素， 模式生物黑腹果蝇的种群。我们的第一个目标是， 果蝇与几个CGD结构，将允许快速评估驱动程序，野生型， 抗性基因型我们的构建体将破坏产生容易识别的隐性基因的靶基因， 表型。此外，它们还将包含产生显性表型的dsRed基因，使我们能够 区分驱动等位基因与不同类型的抗性等位基因，其可能会或可能不会破坏靶基因 功能我们的CGD构建体将被设计为最小化抗性等位基因形成的速率， 通过使用多种gRNA、使用仅雄性生殖系β2-微管蛋白启动子或使用shRNA， 沉默Lig 4-NHEJ机制中的重要组分。在我们的第二个目标中，我们将使用大笼子 数千只苍蝇的种群来研究我们的CGD构建体在2008年的种群动态。 几代人的过程。我们将首先跟踪非驱动性破坏的靶等位基因的频率， 会告诉我们它们的健身成本和笼子里的有效种群大小。然后我们将介绍 将具有我们的CGD构建体的果蝇以低起始频率放入野生型果蝇的笼中。表型将是 在几代中跟踪，抗性等位基因将通过在试验结束时测序来表征。 实验，以确定在蔓延的阻力出现的速度驱动器。所有 实验将在美国农业部检查的节肢动物收容设施内进行，以防止节肢动物逃逸 转基因昆虫在考虑将基因驱动系统用于 野生种群的遗传操纵。从我们的实验中得出的参数将是至关重要的， 在野外模拟这种方法的种群动态，我们的大型笼子系统将提供一个 评估未来进一步抑制耐药性的方法的宝贵资源。 !

Many important human pathogens including those responsible for malaria, dengue, yellow fever, Zika, West Nile fever, and chikungunya are transmitted by mosquitoes. A functioning “gene drive” system could fundamentally change our strategies for the control of these disease vectors, by allowing us to drive genetically engineered alleles into vector populations. The recently developed CRISPR/Cas9 gene drive (CGD) system promises a highly adaptable mechanism for this purpose that works by converting heterozygotes for the driver construct into homozygotes in the germ line. However, it remains unclear how well this mechanism would work in wild populations where genetic heterogeneity and mutation will likely give rise to alleles that are resistant to the drive mechanism. Importantly, such resistance alleles will be produced by the drive itself when cleavage is repaired by nonhomologous end joining (NHEJ). The goal of this proposal is to develop CGD constructs with reduced rate of resistance allele formation and to experimentally quantify the factors that are important to the evolution of resistance alleles in large cage populations of the model organism Drosophila melanogaster. In our first aim, we will engineer transgenic Drosophila with several CGD constructs that will allow for rapid assessment of driver, wild-type, and resistance genotypes. Our constructs will disrupt target genes that produce an easily identifiable recessive phenotype. In addition, they will also contain a dsRed gene producing a dominant phenotype, allowing us to distinguish driver alleles from different types of resistance alleles, which may or may not disrupt target gene function. Our CGD constructs will be designed to minimize the rate of resistance allele formation, either through use of multiple gRNAs, use of the male-germline-only β2-tubulin promoter, or use of a shRNA to silence Lig4 – an essential component in NHEJ machinery. In our second aim, we will use large cage populations of several thousand flies to study the population dynamics of our CGD constructs over the course of several generations. We will first track the frequency of non-driving disrupted target alleles, which will inform us about their fitness costs and the effective population size in our cages. We will then introduce flies with our CGD constructs at low starting frequency into cages of wild-type flies. Phenotypes will be tracked over several generations and resistance alleles will be characterized by sequencing at the end of the experiment to determine the rate at which resistance arose during the spread of the driver. All experiments will be conducted inside a USDA-inspected arthropod containment facility to prevent escape of transgenic insects. There is good reason for caution in considering the use of gene drive systems for genetic manipulation of wild populations. The parameters derived from our experiments will be critical for modeling the population dynamics of such approaches in the wild, and our large cage system will provide a valuable resource for evaluating future approaches engineered to further suppress resistance. !