This report summarises the Year 2 lab project. The project was aimed at studying how the downregulation of a specific gene affects HIV infection. Similarly to last year, term 1 was spent preparing for the project. We began by looking up papers online and reading them to obtain background knowledge. Based on this knowledge, we formulated our hypothesis. Experiments for the project started in term 2 and continued until the end of Year 2.

We applied CRISPR-Cas9 gene editing technique to produce a monoclonal cell line with reduced expression of our target protein. (A gene codes for a protein.) CRISPR-Cas9 can cleave DNA at target sites thanks to guide RNAs (gRNAs). DNA repair mechanisms of a cell randomly repair the cleavage. gRNAs vary depending on experiments, so our project began with designing our gRNAs. We ordered our gRNAs from a manufacturer. In our lab, the gRNAs were inserted into plasmids containing CRISPR-Cas9 gene. The processed plasmids were introduced into TZM_bl cells, commonly used human cells for research in HIV infection, by transfection (using lipofection). After the transfection, the cells can use the introduced plasmids to produce CRISPR-Cas9 protein and our gRNAs. The produced CRISPR-Cas9 proteins performed targeted DNA cleavage guided by gRNAs. We used fluorescence-activated cell sorting (FACS) to identify cells with successful transfection and to isolate these cells. Each isolated cell was grown separately to produce a colony of clone cells.

The target protein expression was measured by Western blot to test the effects of our gene editing. Based on the results, a single colony was selected and used for the rest of the experiments. Some cells from the selected colony were infected with HIV virus-like particles (VLPs). VLPs, resembling viruses but unable to replicate, were used for safety reasons. Then, we investigated the level of infection by measuring the green fluorescent protein (GFP) expression level with a fluorescent microscope. GFP expression level reflected the level of infection, as our cells were supposed to produce GFP upon infection. Additionally, we investigated the level of viral DNA integration into the host cells. HIV is a DNA virus, so it needs to integrate its DNA into the host genome to replicate inside a host cell. To measure the viral DNA integration, the indirect immunofluorescence technique was applied.

In short, this project involved CRISPR-Cas9 to downregulate the target gene; infection with VLPs; measuring the level of infection; and measuring the level of viral DNA integration by immunofluorescence. After completing the project, I needed to submit a report written in a journal-paper format. I refrain from writing the results of my experiments to keep this report concise. Hopefully, this report demonstrates how the level of experiment increased from Year 1. Please refer to my previous report for comparison.

More than half of my undergraduate degree has been completed already. I will endeavour to achieve high grades in Year 3 and contribute back to the Tazaki Foundation.