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oai:www.repository.cam.ac.uk:1810/3527032023-12-22T13:21:22Zcom_1810_224161com_1810_256067col_1810_224162

Enabling the in vitro study of long noncoding RNAs to understand their role in Plasmodium falciparum https://doi.org/10.17863/CAM.98898 Hoshizaki, Johanna Lee, Marcus 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. University of Cambridge 2023-03-31 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/352703 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/7f85076d-38e7-48d6-ad94-7be1b645bcc5/download 1688d7e896db2cf10e1afa7f5bfaf255 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2f78ad16-5908-4f99-8cec-5250666ad900/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ CRISPR gene annotation lncRNAs malaria Plasmodium