Ribozyme Guided CRISPRi in Human- and Rodent-Infectious Plasmodium species

疟疾仍然是当今最大的全球健康问题之一，每年有近50万人死亡，200多万人新感染。这种疾病是由疟原虫寄生虫引起的，疟原虫寄生虫在蚊子载体和它们的脊椎动物宿主（例如人类、小鼠）之间传播。了解寄生虫如何完成这个传播周期，直到对其基因贡献的机械理解，为治疗干预提供了许多机会。然而，分子工具，这将大大有助于理解这些贡献已受到阻碍，需要遗传修饰的寄生虫，以有力地询问每个基因的功能和重要性。这项工作的目的是为啮齿动物感染性模型物种（约氏疟原虫）和人类感染性物种（恶性疟原虫）开发一种灵活而稳健的基因调控系统。在该提议中，将采用范式转换CRISPR/Cas9系统，因为它可以简单地通过引入不同的RNA分子来编程，该RNA分子为感兴趣的基因提供靶向序列。正因为如此，不需要对寄生虫的基因组进行遗传操作，这些研究可以更快地进行，规模也更大。在这项研究中，自催化核酶被用来精确地产生可以对CRISPRi基因调控系统进行编程的单向导RNA（sgRNA）。此外，由于这些sgRNA从其原始RNA分子中裂解出来，因此这些小核酶-向导-核酶单元的聚合物可以简单地化学合成并以“即插即用”形式在一个步骤中插入。另外，任何启动子序列都可以用于表达这些单元，包括可以被调节的、阶段特异性的和具有不同强度的启动子。与该CRISPRi系统的有效活性相关的参数（目标1）将有助于实现对寄生虫传播重要或必需的基因的最大敲低效应。由于这些基因在传播过程中具有强表型，因此可以直接确定CRISPRi的最大敲低是否产生与遗传破坏的寄生虫相同的表型（Aim 2）。总之，这种CRISPRi基因调控系统可以提供灵活，快速和可定制的工具来研究疟原虫寄生虫中的基因功能和重要性。此外，这些研究将不需要对寄生虫的基因组进行遗传修饰，并且可以以大大增加的速度和规模进行。

Malaria remains one of the great global health problems today, with nearly half a million deaths and over two million new infections occurring annually. This disease is caused by Plasmodium parasites, which are transmitted between a mosquito vector and their vertebrate host (e.g. humans, mice). Understanding how the parasite accomplishes this transmission cycle, down to a mechanistic understanding of the contributions of its genes, has provided many opportunities to intervene therapeutically. However, molecular tools that would greatly facilitate understanding these contributions have been hampered by the need to genetically modify the parasite in order to robustly interrogate each gene's functions and importance. The objective of this proposed work is to develop a flexible and robust gene regulation system for both rodent- infectious model species (Plasmodium yoelii) and a human-infectious species (Plasmodium falciparum). In this proposal, the paradigm-shifting CRISPR/Cas9 system will be employed as it is readily programmable simply by introducing a different RNA molecule that provides the targeting sequence for the gene-of-interest. Because of this, no genetic manipulation of the parasite's genome is required and these studies can be carried out much more quickly and in significantly larger scale. In this study, autocatalytic ribozymes are used to precisely produce the single guide RNA (sgRNA) that can program the CRISPRi gene regulation system. Moreover, because these sgRNAs are cleaved out of their original RNA molecule, a polymer of these small Ribozyme-Guide-Ribozyme units can simply be synthesized chemically and inserted in one step in a “plug-and-play” format. Additionally, any promoter sequence can be used to express these units, including promoters that can be regulated, that are stage-specific, and that are of different strengths. The parameters that correlate with efficient activity of this CRISPRi system (Aim 1) will help to achieve the maximal knockdown effect of genes that are important or essential to the transmission of the parasite. Because these genes have strong phenotypes during transmission, it will be straightforward to determine if the maximal knockdown by CRISPRi produces the same phenotype as do the genetically disrupted parasites (Aim 2). Together, this CRISPRi gene regulation system can provide a flexible, rapid, and customizable tool to investigate gene function and importance in Plasmodium parasites. Moreover, these studies will not require genetic modification of the parasite's genome, and can be carried out at a greatly increase pace and scale.