Figure 2. Genetic Strategy to Knockout or Tag Cryptosporidium Thymidine Kinase.

The thymidine locus (tk) can be targeted for knockout or C-terminal epitope tagging. A) Flanks of 50 bp of homology from directly upstream and downstream of the gene target the repair cassette (black) containing Nluc-NeoR (white) to replace the entire tk open reading frame (ORF, red). B) To epitope-tag TK, a repair cassette containing the C-terminus of the gene (red) is cloned fused to the epitope tag (grey). The Nluc-NeoR cassette (white) is expressed under independent regulatory sequences. Flanks of 50 bp of homology (black) target the repair cassette for correct integration and produce a direct fusion of TK with an epitope tag (grey). C) Homology flanks of 50 bp were designed such that the entire TK ORF (red), including the guide RNA sequence (underlined) and the PAM (blue), were replaced with the Nluc-NeoR cassette. D) To fuse TK at the C-terminus with an epitope tag, the C-terminal region of TK (C-term TK, red) was cloned without a stop codon (purple) in frame with an epitope tag (grey). This TK-epitope fusion was cloned upstream of the Nluc-NeoR cassette (white) to generate the repair epitope tagging construct. The epitope tagging construct is flanked by 50 bp of homology (black) from directly upstream of the PAM (blue) and directly downstream of the stop codon (purple). To avoid further targeting of the repair cassette after integration, the PAM is mutated (underlined).