Host Cell Vimentin Restrains Toxoplasma gondii Invasion and Phosphorylation of Vimentin is Partially Regulated by Interaction with TgROP18

He, Cheng; Kong, Ling; Zhou, Lijuan; Xia, Jing; Wei, Haixia; Liu, Min; Peng, Hongjuan

The CRISPR-Cas9 system was adopted to both disrupt rop18 gene and tag rop18 gene with eGFP-FLAG to generate a knockout mutant RH Δrop18 strain and a strain endogenously expressing GFP-FLAG-tagged Rop18 (RH-ROP18-eGFP-FLAG), respectively. To disrupt rop18, we designed a guide RNA (gRNA) to target the 5′ coding region of rop18 (sgROP18), and paired this sgRNA with a PCR amplicon consisting of the DHFR* selectable marker reading frame, flanked with regions homologous to rop18 ( Figure 1A ; Supplementary Material). Parasites were co-transfected with the sgROP18 CRISPR plasmid and homology template, and the recombinant tachyzoites were screened with pyrimethamine and obtained by limiting dilution. Single clones were isolated and verified by PCR to confirm the disruption of rop18 and integration of the DHFR * cassette ( Figure 1B ). To tag endogenous rop18 in T. gondii RH, a Cas9/CRISPR gRNA targeting the C-terminus of rop18 was generated and a homologous template was prepared, as described in methods ( Figure 1C ), and these were cotransfected into RH tachyzoites to generate rop18 with an eGFP-FLAG-tag at its C-terminus. Recombinant clones were confirmed by PCR (using genomic DNA as template) ( Figure 1D ), immunofluorescence (IF) ( Figure 1E ), and western blotting (WB) ( Figure 1F ). A. Schematic of the CRISPR/CAS9 strategy used to inactivate rop18 by insertion of pyrimethamine-resistant DHFR (DHFR*). B. Verification PCR demonstrating homologous integration and gene disruption in a representative clone, compared with RH parental line tachyzoites. C. Schematic illustration of the CRISPR/CAS9 strategy used to tag endogenous ROP18 at the C-terminus with eGFP-FLAG. D. Verification PCR showing correct integration of eGFP-FLAG, SAG1 3′-UTR, and DHFR*. E. IF revealing that eGFP was successfully fused to the C-terminus of endogenous ROP18. F. Demonstration that FLAG tagged ROP18 was expressed by WB with rabbit anti-DDDDK antibody.