Endogenous C-terminal tagging of TcAMT by CRISPR-Cas9.

To obtain the C-terminal tagging of TcAMT, we used the Cas9/pTREX-n vector that we developed for T. cruzi (24) to clone a specific single-guide RNA (sgRNA) sequence targeting the 3′ end of the gene. We cotransfected the 3′-end-tagged sgRNA/Cas9/pTREX-n construct with the specific DNA donor cassette amplified from the pMOTag-4H vector to induce homology-directed repair and to insert a specific tag sequence (3×HA) at the 3′ end of the gene, as described previously using primers 3 to 7 (24).