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. 2020 Feb 15;222(1):126–135. doi: 10.1093/infdis/jiaa072

Figure 6.

Figure 6.

CRISPR/Cas9 strategy used to generate sialic acid binding protein-1 gene (SABP1) knockout (KO) and gene complemented strain. A, Verification of single clonal SABP1-deficient parasite by PCR. The positive KO-PCR1, PCR2 (DNA of ΔSABP1 parasites was used as the template) and negative RH-PCR3 (DNA of RH parasites was used as the template) indicate that the SABP1 gene was replaced by the DHFR cassette. B, SABP1 gene was reintroduced into the genome of the KO strain. PCR of ΔSABP1-C clonal strains (C-PCR4 +, C-PCR5 +, C-PCR7 +, and C-PCR6 −) demonstrated that SABP1 gene was completely inserted at the UPRT locus of the KO genome. C, The expression of SABP1 in RH, ΔSABP1, and ΔSABP1-C parasites analyzed by western blot (20 and 30 μg lysates of each strain) with the SABP1-specific antibody to demonstrate the depletion and complementation of TgSABP1 gene. D, Immunofluorescence assays with SABP1-specific antibodies illustrate that the protein is expressed on the surface of wild-type RH strain (red) and ΔSABP1-C parasites (green), but was not detected on ΔSABP1 parasites. Scale bar, 10 μm. Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.