CRISPR-Cas9 strategy to generate <i>TcIPCS</i> knockout in <i>T</i>. <i>cruzi</i> epimastigotes.

(A) Schematic representation of the Cas9 ribonucleoprotein complexes with the two sgRNAs and their annealing position in the TcIPCS locus. A DNA fragment containing the TcIPCS homologous sequences flanking a Neomycin resistance cassette to allow homologous recombination repair is also shown. After epimastigote transfection, replacement of the TcIPCS coding region by the Neomycin resistance gene is indicated. (B) PCR analysis of wild type and TcIPCS knockout epimastigotes. The annealing position of primers are shown in the schematic representation of one wild type and one disrupted TcIPCS allele with the predicted sizes for the amplicons indicated below the figure. Agarose gel electrophoresis analyses of PCR products obtained with DNA extracted from WT epimastigotes and from two TcIPCS knockout (KO) cloned cell lines, KO4 and KO8 and the indicated primer sets. (C) Relative expression levels of TcIPCS transcripts quantified by RT-qPCR using RNA extracted from WT and two TcIPCS KO cell lines, KO4 and KO8. (D) Negative ion ES-MS lipidomic analysis of WT and TcIPCS KO4 lipid extracts. Spectra show survey scans (600-1000m/z) of WT (upper figure) and KO4 epimastigotes (bottom figure) showing a heterogeneous mixture of PI, IPC, PS and PE. (E) Positive ion ES-MS lipidomic analysis of WT and TcIPCS KO4 lipid extracts. Spectra show survey scans (600-1000m/z), of WT (upper figure) and KO4 epimastigotes (bottom), showing a mixture of PC and PG species.