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

摘要

Author summary We developed an experimental system to study amastigote stage of Trypanosoma cruzi as a proliferable axenic culture. Use of axenic amastigotes allows us to directly introduce exogenous gene into T. cruzi amastigote and select for drug resistant parasite to enrich the transfectants. Our strategy bypasses differentiation steps involved in conventional epimastigote transfection procedure to obtain transgenic amastigotes. Gene knockout can also be performed in amastigote-specific manner, using Cas9-expressing extracellular amastigotes. Drug sensitivity could also be assessed during 1-week axenic growth period. Our method potentially leads to variety of new experimental strategies to make amastigote-stage-specific manipulations and analyses possible.