Generation of the TgIST insertional mutant

Type I (RHku80) and II (Pruku80 and 76KGFP/LUC) were cotransfected with a mixture of the pTOXO_Cas9CRISP::sgIST vector with purified amplicons containing the DHFR cassette flanked by sequences homologous to the sequence targeted by sgIST. These amplicons were generated by PCR amplification of the DHFR cassette using the primers TgIST-DHFR-F and TgIST-DHFR-R and a vector carrying the DHFR cassette as template. To generate a complemented Pruku80 ΔTgIST strain, vector pLIC-P_TgIST-TgIST-HF was coelectroporated with the pMiniHX at 1:10 ratio, followed by selection with mycophenolic acid and xanthine, and cloned by limiting dilution. The resulting strain was named Pruku80 ΔTgIST, pLIC-P_TgIST-TgIST-HF (Table S1).