Purpose: To shed light on the parasiticidal mechanisms of AN13762, we have adapted an original workflow which combines a forward genetic approach based on transcriptome sequencing, computational mutation discovery and CRISPR/Cas9 genome editing in Toxoplasma gondii. Drug-resistant parasites were generated by chemical random mutagenesis. Multiple independent resistant lines were isolated. Single nucleotide variations (SNVs) were identified based on NGS transcriptomic analysis. By focusing on mutations present in coding sequences, we identified a single gene, TgCPSF3, that harbored SNVs leading to amino acid substitutions in each of the 7 drug-resistant lines that were not present in the parental strain. Finally, using CRISPR/Cas9 genome editing we confirmed that the mutations identified confer resistance against AN13762. Overall design: Methods: RNA profiles were generated from HFF cells infected by RH wild-type or AN13762-resistant T. gondii strains. Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). The RNA quality was assessed using TapeStation System (Agilent Technologies, Palo Alto, California, USA). Illumina TruSEQ RNA library prep of selected polyA RNAs and sequencing reagents were used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2 × 125 bp) on the Illumina Hiseq platform and generated at least 40 million reads for each sample.