Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: Proof of concept

• We have adapted the DiCRE system to remove selectable markers in Trypanosoma cruzi.
• We constructed two vectors that can be transferred to different strains.
• We optimized the conditional deletion of DNA fragment from T. cruzi genome.

The scarcity of molecular tools for genetic manipulation is a critical obstacle for functional genomics studies on Trypanosoma cruzi. The current study adapted an inducible site-specific recombination system based on Dimerizable CRE recombinase (DiCRE). Two vectors for stable transfection were created, a first one to express inactive portions of DiCRE recombinase, and a second plasmid containing the loxP sites to test DiCRE activity. After integrating both constructs into the T. cruzi genome, it was shown that DiCRE recombinase can be efficiently used to manipulate its genome by allowing the removal of selectable markers thus generating homogeneous populations. The DiCRE recombinase success allows conditional knockout and the removal of selectable markers without prior parasite modification, which also facilitate the transferring of DiCRE recombinase to different T. cruzi strains.