Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in <i>Trypanosoma cruzi</i>

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.