Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping

Abstract

BackgroundInfection with Trypanosoma cruzi causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disease pathogenesis. Studies are compromised by the complexity of the disease, the long-term nature of the infection, and the fact that parasites are barely detectable during the chronic stage. In addition, functional dissection of T. cruzi biology has been restricted by the limited flexibility of the genetic manipulation technology applicable to this parasite.Methodology/Principal findingsHere, we describe two technical innovations, which will allow the role of the parasite in disease progression to be better assessed. First, we generated a T. cruzi reporter strain that expresses a fusion protein comprising red-shifted luciferase and green fluorescent protein domains. Bioluminescence allows the kinetics of infection to be followed within a single animal, and specific foci of infection to be pinpointed in excised tissues. Fluorescence can then be used to visualise individual parasites in tissue sections to study host-parasite interactions at a cellular level. Using this strategy, we have been routinely able to find individual parasites within chronically infected murine tissues for the first time. The second advance is the incorporation of a streamlined CRISPR/Cas9 functionality into this reporter strain that can facilitate genome editing using a PCR-based approach that does not require DNA cloning. This system allows the rapid generation of null mutants and fluorescently tagged parasites in a background where the in vivo phenotype can be rapidly assessed.Conclusions/SignificanceThe techniques described here will have multiple applications for studying aspects of T. cruzi biology and Chagas disease pathogenesis previously inaccessible to conventional approaches. The reagents and cell lines have been generated as a community resource and are freely available on request.

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…Therefore, the use of two selective markers is probably necessary because in this strategy the sgRNA is transiently expressed (Costa et al. ). For GP72 and mNeonGreen, gene editing was highly efficient.…”

“…; Costa et al. ) and involves the PCR‐amplification of both, a sgRNA template for in vivo transcription and the homology cassettes to induce DSB repair as explained in the T. cruzi section. The PCR‐generated DNA templates are used to transfect parasites constitutively expressing Cas9 and the T7RNAP (Cas9 T7 cells).…”

“…However, it has been shown that only one selective marker is necessary to replace both alleles of a gene when Cas9 is constitutively expressed in T. cruzi (Lander et al 2015). Therefore, the use of two selective markers is probably necessary because in this strategy the sgRNA is transiently expressed (Costa et al 2018). For GP72 and mNeon-Green, gene editing was highly efficient.…”

“…More recently, a genome editing toolkit for kinetoplastids has been successfully used in L. mexicana, L. major, and L. donovani Martel et al 2017). This cloning-free system has also been adapted for gene editing in T. brucei and T. cruzi Costa et al 2018) and involves the PCR-amplification of both, a sgRNA template for in vivo transcription and the homology cassettes to induce DSB repair as explained in the T. cruzi section. The PCR-generated DNA templates are used to transfect parasites constitutively expressing Cas9 and the T7RNAP (Cas9 T7 cells).…”

“…Then, these cell lines were used to generate LmPF16 and TbGPI-PLC (encoding glycosylphosphatidylinositol-specific phospholipase C) gene knockouts, demonstrating the usefulness of the system to perform genome editing in other kinetoplastid species. In addition to the plasmids designed for PCR-generation of template DNAs, Beneke et al (2017) also provided an online tool for primer design using their system, which has been proven to be rapid, reliable, and useful to target single genes and potentially useful for high-throughput genome screening Costa et al 2018;Martel et al 2017).…”