Figure 2

CRISPR/Cas9 mediated endogenous gene tagging with a fluorescent protein. A Schematic illustration for tagging EtMic2 with RFP. Donor fragment for HDR of DSB targets the EtMic2 C-terminal part of the coding sequence (CDS) (red arrow). The left arm (650 bp) is a part of EtMic2 CDS, and the right arm (527 bp) is the 3′ untranslated region of EtMic2. The directions and positions of primers P1 to P4 for PCR identification are indicated by black arrows. B RFP positive sporulated oocysts were observed in 1st generation of EtMic2-RFP. Bar = 5 μm. C Diagnostic PCR demonstrates homologous integration in EtMic2-RFP compared with WT. Predicted size products (808 and 699 bp) were detected after PCR amplification using P1 (5′-CCCTTGATTGCTGTTCGCATCCAT-3′) and P2 (5′-GATCTCGAAGTAGTGGCCGTTCAC-3′) or P3 (5′-GCCGAGGATTTTGAGGTCGTGG-3′) and P4 (5′-TTACCCATGTGGAAGCAACATTGG-3′) primers (A) from genomic DNA of EtMic2-RFP. D Co-localization of the RFP and EtMic2 EtMic2-RFP sporozoites. EtMic2-RFP sporozoites of the 3rd generation were stained with mouse anti-EtMic2 monoclonal antibody and subsequent reaction with FITC-conjugated goat anti-mouse IgG (Proteintech Group). The sporozoites were visualized using a confocal laser scanning microscopy (SP5, Leica, Germany). Sporozoites from wild type (WT) E. tenella serve as control. Bar = 5 μm.