Lentiviral Vector sgRNA Genome Editing on Parental Cells to Inhibit Growth of T. gondii
Conference Year
2024
Abstract
This thesis investigates the applicability of a CRISPR Genome-Wide sgRNA Library to edit the genome of human cells with the aim of uncovering genes that, when deleted, restrict Toxoplasma gondii survival. Initial experiments conducted used HEK 293T cells. A parental cells line was established using Lentiviral transduction to express Cas9. Then, this parental cell lineage was subjected to another Lentiviral Transduction containing the sgRNA library, inducing random single gene edits. The sgRNA lentiviral vectors contained the pLVXS-sgRNA-mCherry-hyg, a plasmid that served as the primary genome for sgRNA replication and enabled successful cell selection methods that were key to obtain edited clones. To test our hypothesis that intracellular factors are crucial for T. gondii infection, we infected the polyclonal population with tachyzoites. As the final selection criterion, clones that were not lysed by T. gondii intracellular growth after 14 days and remaining HEK 293T cells were subjected to genomic extractions and NGS sequencing. The results of the NGS sequencing were used to determine which gRNAs were present in the population of cells refractory to the infection. One of the genes found in the library screen was Necap2, an accessory protein involved in clathrin mediated endocytosis. This gene was selected to be a knockout in a population of A549 cells, which were outsourced and clonally selected to ensure the absence of Necap2 protein. Clones of A549 Necap2 KO cells were infected with T. gondii and subjected to two types of assays: plaque assays and formaldehyde fixation. The current findings of these assays indicate a reduction in T. gondii infection, validating the gene knockout dataset and the Guide-it CRISPR Genome-Wide sgRNA Library System as applicable to human cells genomes to understand host-parasite interactions.
Primary Faculty Mentor Name
Bruno Martorelli Di Genova
Graduate Student Mentors
Gabe Messina, Amber Goerner
Status
Undergraduate
Student College
College of Engineering and Mathematical Sciences
Program/Major
Biomedical Engineering
Primary Research Category
Life Sciences
Lentiviral Vector sgRNA Genome Editing on Parental Cells to Inhibit Growth of T. gondii
This thesis investigates the applicability of a CRISPR Genome-Wide sgRNA Library to edit the genome of human cells with the aim of uncovering genes that, when deleted, restrict Toxoplasma gondii survival. Initial experiments conducted used HEK 293T cells. A parental cells line was established using Lentiviral transduction to express Cas9. Then, this parental cell lineage was subjected to another Lentiviral Transduction containing the sgRNA library, inducing random single gene edits. The sgRNA lentiviral vectors contained the pLVXS-sgRNA-mCherry-hyg, a plasmid that served as the primary genome for sgRNA replication and enabled successful cell selection methods that were key to obtain edited clones. To test our hypothesis that intracellular factors are crucial for T. gondii infection, we infected the polyclonal population with tachyzoites. As the final selection criterion, clones that were not lysed by T. gondii intracellular growth after 14 days and remaining HEK 293T cells were subjected to genomic extractions and NGS sequencing. The results of the NGS sequencing were used to determine which gRNAs were present in the population of cells refractory to the infection. One of the genes found in the library screen was Necap2, an accessory protein involved in clathrin mediated endocytosis. This gene was selected to be a knockout in a population of A549 cells, which were outsourced and clonally selected to ensure the absence of Necap2 protein. Clones of A549 Necap2 KO cells were infected with T. gondii and subjected to two types of assays: plaque assays and formaldehyde fixation. The current findings of these assays indicate a reduction in T. gondii infection, validating the gene knockout dataset and the Guide-it CRISPR Genome-Wide sgRNA Library System as applicable to human cells genomes to understand host-parasite interactions.