CRISPR/Cas9 Plasmid Construction for Toxoplasma Genetics

A complete list of plasmids used or generated in this study is provided in S2 Table. CRISPR/Cas9 plasmids were adapted from the pSAG1:CAS9,U6:sgUPRT plasmid previously generated by our lab [33 (link)]. The guide RNA of the plasmid was modified to target the AAH2 5’ UTR by Q5 mutagenesis (New England Biolabs, Ipswich, MA), creating the plasmid pSAG1:CAS9,U6:sgAAH2. A second guide RNA expression cassette targeting the AAH2 3’ UTR was inserted into the same plasmid backbone by traditional cloning steps to create the CRISPR/Cas9 AAH2 double cut plasmid pSAG1:CAS9,U6:dgAAH2. The same plasmid backbone was similarly adapted to target the AAH1 5’ and 3’ UTRs (pSAG1:CAS9,U6:sgAAH1 and pSAG1:CAS9,U6:dgAAH1). The pSAG1:CAS9,U6:sgUPRT plasmid described previously [33 (link)] was also modified to create a double-cutting CRISPR/Cas9 plasmid targeting the HXGPRT gene pSAG1:CAS9,U6:dgHXGPRT [34 (link)]. Plasmids used to generate the Δaah2 knockout using the HXGPRT selectable marker to replace the gene in the ME49Δhxg::Luc strain [31 (link)], and to restore expression of AAH2 were described previously [24 (link)]. Plasmids used to generate the Δaah1 mutant by replacement with the selectable marker DHFR-Ts, and to complement expression with a cDNA construct targeted to the uracil phosphoribosyl transferase (UPRT) locus, were created using Gibson assembly (New England Biolabs).