Characterization of T. cruzi ΔPGFS mutant clones and PGFS add-backs.

Screening of T. cruzi PGFS knockout clones after CRISPR/Cas9 with sgRNA187 was performed by both PCR and western blot. (A) Schematic representation of PGFS sequence before and after the insertion of donor DNAs with stop codons and XhoI restriction site; (B) The complete PGFS coding sequences (1140 bp) were amplified by PCR. The primers can successfully amplify both the WT sequence and the mutant sequences containing stop codons and the XhoI restriction site. The PCR products were purified and digested with the restriction enzyme XhoI. 3.5 μg of DNA was applied per well. The WT sequence does not have the XhoI site and wasn’t digested. On the other hand, the mutant sequences edited were cleaved by the XhoI restriction enzyme and produced two fragments, one with 216 bp and another with 941 bp. (C) Western blotting shows the deletion of the PGFS gene after CRISPR and also the expression of PGFS in the add-back parasites. The western blots are performed using polyclonal anti-PGFS antibody and monoclonal anti-α-tubulin as a normaliser. The fold-change was calculated using the WT parasites as standards. WT, wild-type; c27 and c29, mutant clones; MW, molecular weight; CN, negative control; bp, base pair; KDa, kiloDalton.