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. 2021 Mar 9;12(2):e03405-20. doi: 10.1128/mBio.03405-20

FIG 5.

FIG 5

Selection of Δins1 parasites and INS1m transgenic parasites in immunocompromised mice. (A) Diagram of the strategy to construct Δins1 transgenic parasites. Construct was designed to replace the INS1 locus with an mCherry and Nluc-P2A-NeoR cassette. The top line shows the targeting construct, the middle line the genomic locus, and the bottom line the successfully targeted transgenic locus. P2A, split peptide; INS1 gRNA, site of guide RNA homology. (B) Diagram of the strategy to construct INS1m active-site mutants. Construct was designed to make an INS1 point mutation in which the active site HLIEH was mutated to AAAAA and added a 3HA tag and Nluc-P2A-NeoR cassette at the C terminus of INS1 (cgd1_1680). The top line shows the targeting construct, the middle line the genomic locus, and the bottom line the successfully targeted transgenic locus. P2A, split peptide; INS1 gRNA, site of guide RNA homology. (C) Selection strategy for obtaining Δins1 or INS1m transgenic parasites. Transfected sporozoites were gavaged into Ifngr1−/− mice treated with 16 g/liter paromomycin in drinking water. A second round of selection was conducted in GKO mice. Each mouse in round two was gavaged with a fecal slurry containing 2 × 104 oocysts collected at 18 dpi of the first round of selection. (D) PCR analysis of Δins1 oocysts obtained from the second round of amplification. WT, wild type. KO, Δins1 parasite. The product 5′ Ins is specific for the 5′ CRISPR targeting site of the Δins1 parasite. The product 3′ Ins is specific for the 3′ CRISPR targeting site of Δins1 parasite. The product ORF1 detects a 2,703-bp fragment of the INS1 open reading frame. The product ORF2 detects a 468-bp fragment of the INS1 open reading frame. (E) PCR analysis of INS1m oocysts obtained from the second round of amplification. WT, wild type. mu, INS1m parasite. The product 5′ Ins is specific for the 5′ CRISPR targeting site of INS1m. The product 3′ Ins is specific for the 3′ CRISPR targeting site of INS1m. Control, product is specific to the INS3 locus. (F) Sequence electropherogram of PCR products from native INS1 (top) and active-site mutant INS1m (bottom) transgenic parasites. Native, the amino acid and nucleotide sequence of the active site in wild-type INS1 parasite. INS1m, the amino acid and nucleotide sequence of the active site in INS1m parasite.