Trypanosoma cruzi is a protozoan parasite of humans and animals, affecting 10 to 20 million people and innumerable animals, primarily in the Americas. Despite being the largest cause of infection-induced heart disease worldwide, even among the neglected tropical diseases (NTDs) T. cruzi is considered one of the least well understood and understudied. The genetic complexity of T. cruzi as well as the limited set of efficient techniques for genome engineering contribute significantly to the relative lack of progress in and understanding of this pathogen. Here, we adapted the CRISPR-Cas9 system for the genetic engineering of T. cruzi , demonstrating rapid and efficient knockout of multiple endogenous genes, including essential genes. We observed that in the absence of a template, repair of the Cas9-induced double-stranded breaks (DSBs) in T. cruzi occurs exclusively by microhomology-mediated end joining (MMEJ) with various-sized deletions. When a template for DNA repair is provided, DSB repair by homologous recombination is achieved at an efficiency several orders of magnitude higher than that in the absence of CRISPR-Cas9-induced DSBs. We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations. Lastly, we show that Cas9 can mediate disruption of its own coding sequence, rescuing a growth defect in stable Cas9-expressing parasites. These results establish a powerful new tool for the analysis of gene functions in T. cruzi , enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome. IMPORTANCE Trypanosoma cruzi , the causative agent of human Chagas disease, is the leading worldwide cause of infectious myocarditis. Diagnostics for the infection are relatively poor, treatment options are limited and of variable effectiveness, and suitable vaccines are nonexistent. The T. cruzi genome is replete with genes of unknown function and greatly expanded gene families with hundreds of members. The absence of facile genetic engineering tools, including RNA interference, for T. cruzi has prevented elucidation of gene and gene family function and the development of better infection prevention and control measures. In this study, we demonstrate that the CRISPR-Cas9 system is a versatile and powerful tool for genome manipulations in T. cruzi , bringing new opportunities for unraveling the functions of previously uncharacterized genes and how this human pathogen engages its large families of genes encoding surface proteins to interact with human and animal hosts.

Xue and colleagues show that Runx3−/− CD8+ T effector cells aberrantly upregulate genes characteristic of TFH cell lineage and exhibit impaired induction of cytotoxic molecules. Activated CD8+ T cells differentiate into cytotoxic effector (TEFF) cells that eliminate target cells. How TEFF cell identity is established and maintained is not fully understood. We found that Runx3 deficiency limited clonal expansion and impaired upregulation of cytotoxic molecules in TEFF cells. Runx3-deficient CD8+ TEFF cells aberrantly upregulated genes characteristic of follicular helper T (TFH) cell lineage, including Bcl6, Tcf7 and Cxcr5. Mechanistically, the Runx3-CBFβ transcription factor complex deployed H3K27me3 to Bcl6 and Tcf7 genes to suppress the TFH program. Ablating Tcf7 in Runx3-deficient CD8+ TEFF cells prevented the upregulation of TFH genes and ameliorated their defective induction of cytotoxic genes. As such, Runx3-mediated Tcf7 repression coordinately enforced acquisition of cytotoxic functions and protected the cytotoxic lineage integrity by preventing TFH-lineage deviation.

Significance: Inflammatory responses to viral infections must be optimized to clear the pathogen without tissue damage. Inflammasomes comprise an important component of the innate immune response. Inflammasome activity must be carefully controlled to prevent a hyperinflammatory response, especially in brain infections. Here we identify a host factor, PYDC3, that is dependent upon prostaglandin D2 (PGD ₂ ) and IFN-I signaling and is required to modulate inflammasome activation. After infection, inflammasome activation and expression of a downstream proinflammatory cytokine, IL-1β, were increased in mice deficient in PGD ₂ signaling, decreasing survival. Excess mortality was reversed by IL-1β receptor blockade. These results define a consequence of prostaglandin signaling and shed light on prostaglandin–inflammasome interactions, which modulate excessive inflammation and tissue damage in the virus-infected brain.