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

疟疾仍然是当今严重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引起的，这种寄生虫在蚊子媒介和它们的脊椎动物宿主(如人、鼠)之间传播。了解寄生虫是如何完成这一传播周期的，包括对其基因贡献的机械性理解，为进行治疗干预提供了许多机会。然而，由于需要对寄生虫进行基因改造以有力地询问每个基因的功能和重要性，极大地有助于理解这些贡献的分子工具受到了阻碍。这项拟议工作的目的是为啮齿动物感染的模式物种(约氏疟原虫)和人类感染的物种(恶性疟原虫)建立一个灵活而健壮的基因调控系统。在这个提议中，将使用范式转换CRISPR/Cas9系统，因为它很容易编程，只需引入不同的RNA分子，为目标基因提供靶向序列。正因为如此，不需要对寄生虫的基因组进行基因操作，这些研究可以更快、更大规模地进行。在这项研究中，自催化核酶被用来精确地产生能够编程CRISPRi基因调控系统的单引导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Ribozyme-Guide-Ribozyme单元的聚合物可以简单地通过化学方法合成并以即插即用的形式在一步中插入。此外，任何启动子序列都可以用来表达这些单位，包括可调控的、阶段特异性的和不同强度的启动子。与CRISPRi系统(目标1)的有效活性相关的参数将有助于实现对寄生虫传播重要或必要的基因的最大击倒效应。由于这些基因在传播过程中具有很强的表型，因此很容易确定CRISPRi的最大敲除是否会产生与遗传中断的寄生虫相同的表型(目标2)。总之，这种CRISPRi基因调控系统可以提供一种灵活、快速和可定制的工具来研究疟原虫的基因功能和重要性。此外，这些研究不需要对寄生虫的基因组进行基因改造，而且可以以极大的速度和规模进行。

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.