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

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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.