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
A. Map of pLEW13-Cas9 showing the construct and the integration of linearised DNA into the T. cruzi genome via the tubulin locus (Methods). B. Western blots showing expression of the Cas9 gene. Wild type T. cruzi CL-Brener before (lane 1) and after (lane 2) transfection with pLEW13-Cas9. Right hand panel; T. cruzi CL-Luc::Neon reporter strain before (lane 3) and after (lane 4) transfection. The blot was probed with anti-Cas9 monoclonal 7A9 (Merck). C. Cas9 has no effect on growth when expressed from the tubulin array. Growth curves comparing both parental lines with their Cas9 expressing counterparts. Growth assays were performed in triplicate; the error bars represent standard deviation.