Enabling the in vitro study of long noncoding RNAs to understand their role in Plasmodium falciparum
Long noncoding RNAs (lncRNAs) have been identified in *Plasmodium falciparum*, the parasitic cause for life-threatening malaria, yet their role remains largely undiscovered. Through interactions with nucleic acids and proteins, lncRNAs can modulate gene expression at the transcriptional, post-transcriptional, translational, and post-translational levels. Determining the role of lncRNAs in the regulation of the *P. falciparum* transcriptome and proteome is imperative to further our understanding of gene regulation in the parasite. The characterisation of *P. falciparum* lncRNAs has been hindered by an incomplete annotation and the absence of disruption methods that together would permit high-throughput systematic knockdown of lncRNAs. During my PhD, I addressed these challenges to enable the study of *P. falciparum* lncRNAs *in vitro*. I generated a high-quality lncRNA annotation using manual curation of sequencing data generated at the Sanger Institute, along with supportive datasets from the literature. I evaluated CRISPR-based approaches for *in vitro* disruption of lncRNAs including gene knockout, knockdown, and interference. CRISPR-associated enzymes were explored including commonly used DNA-cutting enzymes (Cas9), inactivated enzymes to block transcription (dCpf1) and enzymes that target RNA directly (Cas13), the latter of which had not been applied to *Plasmodium*. Furthermore, I implemented these tools to demonstrate the feasibility of lncRNA studies in *P. falciparum*. I interrogated a set of lncRNAs that were selected based on predicted biological significance and targetability using dCpf1. LncRNA-depleted parasites were phenotypically characterised by assessing changes in fitness, drug resistance, gametocytogenesis and expression. I identified potential roles for specific lncRNAs in drug resistance and gametocytogenesis. By developing bioinformatics and molecular tools, this work enables future studies elucidating the specific roles of lncRNAs in *P. falciparum*. Understanding the transcriptome and gene regulation will inform the development of novel interventions for the control and eradication of malaria, which remains a serious global health concern.
CRISPR, gene annotation, lncRNAs, malaria, Plasmodium
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