Development, characterization and application of CRISPR/Cas9 gene drive technologies and related active genetic elements to benefit research and society at large

项目摘要 2014年12月18日上午，我早早地来到实验室，检查我的实验--什么？ 最终成为第一个证明诱变连锁反应（MCR）作为一种高效的 水果中基于CRISPR/Cas的基因驱动系统[1].我后来建立了一个类似的，虽然更复杂，MCR 在蚊子中构建，这是与詹姆斯小组（UCI）合作测试的[2]。的情况一样 蚊子MCR通过生殖系以极高的效率（99.5%）繁殖。期间 在这个过程中，我的顾问Ethan Bier和我将“主动遗传学”的概念扩展到一个遗传家族， 主动将自身复制到伴随染色体上的元件（如MCR）。这些元素 绕过孟德尔遗传的限制，从而可能克服目前的限制， 实验室实验基因驱动系统可用于对抗病媒传播的疾病， 全球公共卫生（例如，消灭疟疾），以及恢复当地生态系统（例如，抑制侵入性 种群）。虽然我对未来在不同领域的应用感兴趣，但在获奖期间， 将致力于深化对果树活性遗传因子作用机制的认识。 在这里，我建议在果蝇中建立和描述三类活跃的遗传学特征。 元件：（1）完整的MCR基因驱动，（2）分裂，“反互补MCR”，一种替代方案，可以提供 在野生环境中进行群体修饰时的优势，（3）阻止，限制或 逆转基于Cas9的基因驱动在野外的传播。 1)我将研究我们现有的基因驱动技术的非凡效率的基础，并完善其 未来应用领域的功能。将开发基于MCR技术的几种基因驱动器 在水果蛋糕里。将识别不同的调控区域以驱动Cas9核酸酶在细胞中的表达。 在发育过程中最有效的时间，同时确保其限制在生殖细胞。 2)我将构建和测试反式互补MCR，其中两个主要MCR组分（Cas9和Cas9） gRNA）被分成两个单独的转基因构建体。每个组件单独不会生成 遗传偏见;只有当这些因素结合起来，才能重建基因驱动的安排。这 技术可以用于人口抑制计划，其中全基因驱动，故意影响 否则，将实验室人群扩增到 需要现场释放。 3)我将开发逆转构建体，可以抵消基于Cas9的基因驱动构建体在细胞中的传播。 人口我将测试两种不同类型的这种结构：第一种是通过剪切和替换 基因驱动器在相同的位点，它被插入;第二种类型是位于不同的位置， 基因组，但携带能够利用Cas9蛋白破坏Cas9基因本身的向导RNA。

PROJECT SUMMARY On the morning of December 18th 2014, I arrived in the laboratory early to check on my experiment — what ended up being the first proof that the Mutagenic Chain Reaction (MCR) functioned as a highly efficient CRISPR/Cas-based gene-drive system in fruit flies [1]. I later built a similar, albeit more complex, MCR construct in mosquitoes, that was tested in collaboration with the James group (UCI) [2]. As was the case for the fruit fly element, the mosquito MCR propagates with exceptional efficiency (99.5%) via the germline. During this process, my advisor Ethan Bier and I expanded the concept of `active genetics' [4] to a family of genetic elements that actively copy themselves onto the companion chromosome (as in the MCR). These elements bypass the constraints of Mendelian inheritance, thereby potentially overcoming current limitations in laboratory experiments. Gene drive systems can be used to combat vector-borne diseases thereby benefiting global public health (e.g., malaria eradication), as well as to restore native ecosystems (e.g., suppress invasive species populations). Although I am interested in future applications in diverse fields, during the award period I will focus on deepening the knowledge on the mechanism of action of active genetic elements in the fruit fly. Here I propose to build and characterize in Drosophila melanogaster three categories of active genetic elements: (1) Full MCR-gene drives, (2) Split, “transcomplementing-MCR”, an alternative that could offer advantages when performing population modification in the wild, (3) Reversal constructs to stop, limit or reverse the spread of a Cas9-based gene drive in the wild. 1) I will examine the basis for the extraordinary efficiency of our existing gene drive technology and refine its functionality for future field applications. Several gene drives, based on MCR technology, will be developed in the fruit fly. Different regulatory regions will be identified to drive the expression of the Cas9 nuclease in the most effective time during development while assuring its restriction to germline cells. 2) I will build and test trans-complementing-MCRs in which the two primary MCR components (Cas9 and gRNAs) are split in two separate transgenic constructs. Each component individually would not generate inheritance bias; only when combined will these elements reconstitute a gene drive arrangement. This technology could be used in population suppression schemes where a full gene drive, purposely affecting fitness, would otherwise render problematic the amplification of the laboratory population to the levels necessary for field release. 3) I will develop reversal constructs that can counteract the spread of a Cas9-based gene drive construct in a population. I will test two different types of such constructs: the first one acts by cutting out and replacing the gene drive at the same locus at which it is inserted; the second type is located in a different location in the genome, but carries guide-RNAs able to exploit the Cas9 protein to disrupt the Cas9 gene itself.