Developing CRISPR/Cas9 for genome-wide gene editing in the human pathogen Trypanosoma cruzi
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The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, can infect nearly all mammals and infections are usually lifelong if not treated. The mammalian host’s CD8+ T cell response is crucial to controlling this intracellular parasite. However, the development of the host CD8+ T cell response is significantly delayed compared to that against other pathogens. A significant proportion of host CD8+ T cells in T. cruzi infection is specific for peptides derived from the trans-sialidase (TcTS) gene family. The reference genome of T. cruzi encodes 3209 TcTS genes and it is hypothesized that this large TcTS gene family repertoire aids in immune evasion. One way to test this hypothesis is to create a TcTS-knockdown T. cruzi line. Here, I aimed to build that foundation by knocking-down of the expression of TcTS genes and as well as knocking-out the coding capacity of silent TcTS, so as to impede the restoration of TcTS expression by recombination. To accomplish this, I adapted the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-associated) system for use in T. cruzi. CRISPR/Cas9 system is a genome editing tool adapted from bacteria. Using this system, I demonstrated rapid and efficient gene knockout in T. cruzi, sequence editing using DNA donor template and the ability to knockdown a gene family of 65 members. I next developed a computation tool: the eukaryotic pathogen gRNA design tool (EuPaGDT, http://grna.ctegd.uga.edu) capable of designing CRISPR/Cas9 gRNA sequences targeting genes at a genome-wide scale. EuPaGDT’s unique algorithm allows on-target search, which enumerates all the gene family members that a gRNA targets. Using this tool, I designed 21 gRNAs, that when combined, target more than 87% of TcTS capable of producing peptides and 73% of TcTS at least 30% of full gene length. I sequentially delivered the 21 gRNAs in complex with Cas9 protein and associated repair templates to T. cruzi. The resulting T. cruzi parasites population showed a 55% decrease in TcTS-binding fetuin staining. Sequencing of the gene editing sites in TcTS revealed successful editing of TcTS at multiple sites.