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

疟疾仍然是当今全球最大的健康问题之一，每年有近50万人死亡，200多万新感染病例。这种疾病是由疟原虫引起的，在蚊子媒介和它们的脊椎动物宿主（如人类、老鼠）之间传播。了解寄生虫如何完成这种传播周期，直至对其基因贡献的机制理解，为干预治疗提供了许多机会。然而，分子工具将极大地促进理解这些贡献，但由于需要对寄生虫进行遗传修饰，以强有力地询问每个基因的功能和重要性，这些工具受到了阻碍。本研究的目的是为啮齿动物感染模型种（约氏疟原虫）和人类感染物种（恶性疟原虫）建立一个灵活而强大的基因调控系统。在这个提议中，范式转换的CRISPR/Cas9系统将被采用，因为它很容易通过引入一个不同的RNA分子来提供目标基因的靶向序列来编程。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、更大规模地进行。在本研究中，使用自催化核酶来精确产生可编程CRISPRi基因调控系统的单导RNA （sgRNA）。此外，由于这些sgrna是从它们原来的RNA分子中切割出来的，这些小的核酶-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”的形式一步插入。此外，任何启动子序列都可以用来表达这些单位，包括可调节的启动子、特定阶段的启动子和不同强度的启动子。与该CRISPRi系统有效活性相关的参数（Aim 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很强的表型，因此可以直接确定CRISPRi的最大敲除是否与基因被破坏的寄生虫产生相同的表型（目的2）。总之，这个CRISPRi基因调控系统可以为研究疟原虫的基因功能和重要性提供一个灵活、快速和可定制的工具。此外，这些研究将不需要对寄生虫的基因组进行基因改造，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.