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.